CN101970470B - Novel bacillus thuringiensis gene with coleopteran activity - Google Patents

Abstract

The invention provides nucleic acids, and variants and fragments thereof, obtained from strains of Bacillus thuringiensis encoding polypeptides having pesticidal activity against insect pests, including Coleoptera. Particular embodiments of the invention provide isolated nucleic acids encoding pesticidal proteins, pesticidal compositions, DNA constructs, and transformed microorganisms and plants comprising a nucleic acid of the embodiments. These compositions find use in methods for controlling pests, especially plant pests.

Description

The new Bacillus thuringiensis Genes with coleopteran-active

Invention field

The present invention relates to the naturally occurring nucleic acid and the recombinant nucleic acid that from new bacillus thuringiensis (Bacillus thuringiensis) gene, obtain, described genes encoding be take the insecticidal peptide that pest-resistant harmful insecticidal activity is feature.The compositions and methods of the invention are controlled plant epidemic disease evil with disclosed nucleic acid and coded insecticidal peptide thereof.

Background of invention

Insect pest is the principal element of world's crop loss.For example, west corn rootworm (Westerncorn rootworm) feed, the infringement of black cutworm or European corn borer infringement can destroy agricultural producer economically.The relevant crop loss of insect pest that only bring with sweet corn in European corn borer attack field just reaches annual 1000000000 dollars on infringement and control overhead.

Traditionally, the main method that affects insect pest colony is application phosphoramidite chemical insecticide.Yet human consumer is the same with the adjusting person of government to be more and more concerned about and to produce and use the environmental hazard that synthetic chemical insecticide is relevant.Adjusting person has forbidden for this care or has limited and use some more dangerous sterilants.Therefore, the alternative sterilant of exploitation has substantive interests.

The microorganism agent of use such as fungi, bacterium or other insect species carries out biology control to insect pest important in agricultural, the alternative route that this provides environmentally friendly and had commercial appeal for synthetic chemical insecticide.In general, use biotic pesticide to pollute with the risk of environmental hazard lower, and biotic pesticide provide stronger target specificity with respect to the character of traditional phosphoramidite chemical insecticide.In addition, the production cost of biotic pesticide is conventionally lower, and thereby improves the economic return of the farm crop of a large amount of kinds.

The microorganism of some kind of known bacillus has the insecticidal activity of anti-wide spectrum insect pest, and described insect pest comprises lepidopteran (Lepidoptera), Diptera (Diptera), Coleoptera (Coleoptera), Hemiptera (Hemiptera) and other.Bacillus thuringiensis (Bt) and beetle genus bacillus (Bacillus papilliae) belong to the most successfully biocontrol agent found so far.Entomopathogenic is also owing to bacillus larvae (B.larvae), slow disease bacilli (B. lentimorbus), Bacillus sphaericus (B.sphaericus) (Harwook, ed., ((1989) Bacillus (genus bacillus) (Plenum Press), 306) and bacillus cereus (B.cereus) (WO 96/10083) bacterial strain.Although also isolate insecticidal proteins from the genus bacillus of vegetative growth phase, insecticidal activity looks and concentrates on parasporal crystal protein inclusion body.Several genes of separated these insecticidal proteins of also sign coding (referring to, for example, United States Patent (USP) the 5th, 366, No. 892 and the 5th, 840, No. 868).

Microorganism insecticide, particularly, available from Bacillus strain those, plays an important role in agricultural as the harmful substitute of controlling of chemical epidemic disease.Recently, agri-scientific research worker produces the insecticidal proteins from genus bacillus by genetic engineering modified crop plants, has developed the crop plants of the insect-resistant with enhancing.For example, corn and vegetable lamb by genetically engineered with produce the separated insecticidal proteins from Bt bacterial strain (referring to, for example, Aronson (2002) CellMol.Life Sci.59 (3): 417-425; Schnepf et al. (1998) Microbiol Mol Biol Rev.62 (3): 775-806).These genetically engineered farm crop are widely used in american agriculture now, and the environmental friendliness substitute of traditional insect control is provided to peasant.In addition to American farmers, sell through potato genetic engineering modified and Cry toxin that comprise desinsection.

Still need to have pest-resistant evil widely and kill the new Bt toxalbumin of insect active, for example the activated toxin of insect tool to more kinds in Coleoptera.In addition, still need to have the biotic pesticide of anti-more insect pest activity and there are the biotic pesticide that insect active is killed in improvement.

Summary of the invention

The invention provides and affect the composition of insect pest and method.More specifically, embodiments of the present invention relate to the method that affects insect, and described method utilization coding kills the nucleotide sequence of insect peptide, thereby produce microorganism and the plant of expressing the conversion of killing insect polypeptide in embodiment.These insects comprise the upper important insect of agricultural, such as, for example, west corn rootworm, for example Zea mays root firefly chrysomelid (Diabrotica virgifera LeConte).In some embodiments, this nucleotide sequence coded polypeptide to belonging at least one insect of Coleoptera with pesticidal.

Embodiment provides nucleic acid and fragment and its variant, and its coding has the polypeptide (for example, the SEQ ID NO:1 of coding SEQ ID NO:2) of pest-resistant harmful insecticidal activity.The nucleotide sequence coded new insect peptide that kills of the wild-type of embodiment (for example, naturally occurring), described nucleotide sequence obtains from Bt.Embodiment also provides disclosed encoding human to learn fragment and the variant of the nucleotide sequence of active (for example, killing insect) polypeptide.

Embodiment also provides separated desinsection (for example, killing insect) polypeptide, and its naturally occurring nucleic acid by embodiment or modified (for example, sudden change or through operation) nucleic acid are coded.In particular instance, the insecticidal proteins of embodiment comprises the fragment of full-length proteins and polypeptide, and it produces the nucleic acid from sudden change, and described nucleic acid is through designing specific amino acid sequence to be introduced to the polypeptide of embodiment.In specific implementations, described polypeptide with respect to its derived from naturally occurring polypeptide there is the insecticidal activity of enhancing.

The nucleic acid of embodiment can also for generation of genetically modified (for example, transform) unifacial leaf or dicotyledons, described plant is characterised in that the constructs that genome comprises at least one stable integration, the encoding sequence that described construct comprises embodiment, described encoding sequence is operably connected with the promotor that drives coded insecticidal peptide to express.Therefore, vegetable cell, plant tissue, plant and the seed thereof of conversion are also provided.

In specific implementations, can produce the plant of conversion with improving the nucleic acid of expressing through optimization in host plant.For example, a kind of insecticidal peptide that can retroversion (backtranslate) embodiment, thereby produce the nucleic acid be included as the codon of expressing and optimizing in specific host, described specific host is for example such as the crop plants of corn (Zea mays) plant.The plant of this conversion (for example, dicotyledonous or monocotyledons) is expressed encoding sequence, thereby causes producing insecticidal peptide, and gives the insect-resistant that described plant increases.Some embodiments provide the transgenic plant of expressing insecticidal peptide, and described insecticidal peptide is for affecting the method for various insect pests.

Embodiment also comprises the desinsection that kills insect polypeptide or the insecticidal mixtures that contains embodiment, and can optionally comprise the other insect peptide that kills.Embodiment comprises said composition is applied to insect pest environment, thereby affects insect pest.

Detailed Description Of The Invention

Embodiments of the present invention relate to affects insect pest, particularly composition and the method for plant epidemic disease evil.More specifically, the nucleotide sequence that the separated nucleic acid of embodiment and fragment thereof and its variant comprise coded insect-killing polypeptide (for example albumen).Disclosed insecticidal proteins has biologic activity (for example pesticidal) to the insect pest such as, but not limited to Coleoptera insect pest.Object insect pest includes but not limited to west corn rootworm (Zea mays root firefly is chrysomelid).

The composition of embodiment comprises separated nucleic acid and fragment and its variant, and its coded insect-killing polypeptide, the expression cassette of nucleotide sequence that comprises embodiment are, insecticidal proteins and the insect-killing composition of separation.Some embodiments provide modified insecticidal peptide, and described polypeptide is characterised in that the anti-Coleoptera that the insecticidal activity with respect to corresponding wild-type protein has an improvement kills insect active.Embodiment also provides plant and the microorganism transforming with these new nucleic acid, and relates to this nucleic acid, insect-killing composition, inverting biological body and product thereof for affecting the method for insect pest.

The nucleic acid of embodiment and nucleotide sequence can be for transforming any organism, thereby produce coded insecticidal proteins.The method providing relates to this inverting biological body for impact or control plant epidemic disease evil.The nucleic acid of embodiment and nucleotide sequence can also be for transforming organoid (McBride et al. (1995) the Biotechnology 13:362-365 such as chloroplast(id); With Kota et al. (1999) Proc.Natl.Acad.Sci.USA 96:1840-1845).

Embodiment also relates to naturally occurring encoding sequence fragment and the variant of identification code biologic activity insecticidal proteins.The method of the insect pest that the nucleotide sequence of embodiment is directly used in affects epidemic disease evil, particularly do harm to such as Coleoptera epidemic disease.Therefore, embodiment provides the novel method that affects insect pest, and described method does not rely on the use of traditional synthetic chemical insecticide.Embodiment relates to the gene of finding naturally occurring, biodegradable sterilant and encoding them.

Embodiment also provides same encoding human to learn naturally occurring encoding sequence fragment and the variant of active (for example, desinsection) polypeptide.The nucleic acid of embodiment comprises through optimizing nucleic acid or the nucleotide sequence for the cell expressing of specific organism, for example use favorite plant codon, take to have and strengthen the nucleotide sequence that the polypeptid acid sequence of insecticidal activity is basis retroversion (being reverse translation).Embodiment also provides sudden change, and character polypeptide improvement or that change of embodiment is given in described sudden change.Referring to, for example, the U. S. application the 10/606th that submit to common 25 days unsettled June in 2003, the 10/746th of No. 320 and submission on December 24th, 2003, No. 914.

In description subsequently, a large amount of terms have been widely used.For promoting the understanding to embodiment, provide to give a definition.

Can use the acceptable form representation unit of SI, prefix and symbol.Unless otherwise stated, respectively, nucleic acid is write from left to right with 5 ' to 3 ' direction; Aminoacid sequence is write from left to right with amino to carboxyl direction.Digital scope comprises the numeral that limits this scope.Amino acid herein can be with it letter character of recommending of trigram symbol known to conventionally or IUPAC-IUB biochemical nomenclature commission represent.Similarly, can use the single-letter representation Nucleotide of conventionally accepting.With reference to specification sheets integral body, define more fully term defined above.

As used herein, " nucleic acid " comprises deoxyribonucleotide or the ribonucleotide polymer that relates to strand or double chain form, unless and separately restricted, comprise there is natural nucleotide essential property known analogue (for example, peptide nucleic acid(PNA)), described analogue is to hybridize with mode like naturally occurring ucleotides and single-chain nucleic acid.

As used herein, term " coding " or " coded " during for the context of specific nucleic acid, refer to that this nucleic acid comprises the essential information that instructs this nucleotide sequence to translate into specific protein.The information of subrepresentation proteins encoded accesses to your password.The nucleic acid of proteins encoded can comprise and is positioned at the non-translated sequence (for example, intron) in this translated nucleic acid district or can lacks such non-translated sequence between two parties (for example,, as in cDNA).

As used herein, " full length sequence " that relates to specific polynucleotide or its coded albumen refers to have whole nucleotide sequence or the whole aminoacid sequence of natural (non-synthetic) endogenous sequence.The total length of this specific protein of total length polynucleotide encoding, catalytic activity form.

As used herein, for the contextual term of nucleotides sequence column direction " antisense ", relate to two times of polynucleotide sequences, described polynucleotide sequence is operably connected with promotor to transcribe the direction of this antisense strand.This antisense strand and endogenous transcription product are fully complementary, so that the translation of this endogenous transcription product is conventionally suppressed.Thereby during for the context of specific nucleotide sequence, this term relates to this with reference to the complementary strand of transcription product by term " antisense ".

Use interchangeably term " polypeptide ", " peptide " and " albumen " herein, to refer to the polymer of amino-acid residue.This term is for amino acid polymer, and wherein one or more amino-acid residues are corresponding naturally occurring amino acid whose artificial chemistry analogues.This term is also for naturally occurring amino acid polymer.

Use interchangeably term " residue " or " amino-acid residue " or " amino acid " herein, to refer to be merged in the amino acid of albumen, polypeptide or peptide (general designation " albumen ").Amino acid can be naturally occurring amino acid, unless and separately restricted, can comprise the known analogue of natural amino acid, described analogue can be to work with the mode of naturally occurring amino acid similarity.

Can or use standard molecular biological technique to prepare the polypeptide of embodiment from nucleic acid disclosed herein.For example, can be by express the recombinant nucleic acid of embodiment in suitable host cell, or the combination of (ex vivo) method in junctor between selectively passing through, prepare the albumen of embodiment.

As used herein, can use interchangeably term " separation " and " purifying ", to relate to nucleic acid or polypeptide or its biologic activity part, it does not contain as the component of conventionally following or react on this nucleic acid or polypeptide of being found in its naturally occurring environment substantially or in essence.Thereby, while producing nucleic acid separated or purifying or polypeptide with recombinant technology, nucleic acid or polypeptide separated or purifying do not basically contain other cellular material or substratum, or when nucleic acid chemosynthesis separation or purifying or polypeptide, do not basically contain precursor or other chemical.

" separated " nucleic acid be not conventionally contained in this nucleic acid institute derived from the genomic dna of organism in natural flank in the sequence of this nucleic acid (being positioned at this nucleic acid 5 ' and the 3 ' sequence of holding) (for example, such as, the sequence of proteins encoded).For example, in various embodiments, separated nucleic acid can comprise and be less than about 5kb, 4kb, 3kb, 2kb, 1kb, the nucleotide sequence of 0.5kb or 0.1kb, described nucleotides sequence be listed in this nucleic acid derived from the genomic dna of cell in natural flank in this nucleic acid.

As used herein, by term " separated " or " purifying " when relating to the polypeptide of embodiment, referring to that separated albumen does not basically contain cellular material and comprises to have is less than approximately 30%, 20%, the albumen prepared product of the contaminating protein of 10% or 5% (dry weight basis).When being recombinantly produced the albumen of embodiment or its biologic activity part, substratum representative is less than approximately 30%, 20%, the precursor of 10% or 5% (dry weight basis) or non-target protein chemical.

Run through application documents, word " is comprised to (comprising) " or be understood as prompting such as the variant of " comprising (comprises) " or " comprising (comprising) " and comprise alleged element, integer or step, or the group of element, integer or step, but not get rid of any other element, integer or step, or the group of element, integer or step.

As used herein, term " affects insect pest " and refers to the influential change to insect feed, growth and/or the behavior of any etap, includes but not limited to kill insects, growth-delaying, obstruction reproductive performance, antifeedant activity etc.

As used herein, by term " insecticidal activity " and " killing insect active " synonymously use to refer to organism or material (such as, albumen for example) activity, its measurement can be lost weight through but not limited to Mortality of insect, insect, pest repelling (repellency) and feed and expose suitably long-time after other behavior and the physical change of insect.Thereby organism or the material with insecticidal activity adversely affect at least one measurable insect health parameters.For example, " insecticidal proteins " is self show or show the albumen of insecticidal activity with other protein combination.

As used herein, term " insecticidal effective dose " refers to be present in the material with insecticidal activity of insect environment or the amount of organism.For each material or organism, by experience, determine the insecticidal effective dose of affected each insect in specific environment.Similarly, when insect is insect, " killing insect significant quantity " can be used in reference to " insecticidal effective dose ".

As used herein, term " recombined engineering " or " through engineering approaches " refer to utilize recombinant DNA technology to introduce the change of (for example through engineering approaches) protein structure, and this is based on to the understanding of albumen mechanism of action and to being introduced into, being lacked or substituted amino acid whose consideration.

As used herein, term " sudden change nucleotide sequence " or " sudden change " or " nucleotide sequence of mutagenesis " refer to through mutagenesis or change and the nucleotide sequence that comprises one or more nucleotide residues (for example base pair), and described nucleotide residue is not present in corresponding wild-type sequence.Such mutagenesis or one or more interpolations, disappearance or the replacement or the replacement institute that change by nucleic acid residue form.When adding, removing or replace the amino acid in proteolysis site and prepare sudden change, such interpolation, removing or replace can be in this proteolysis site motif or contiguous this motif, as long as completed the object (as long as having changed the proteolysis in this site) of sudden change.

Mutant nucleotide sequence can kill insect toxins by encoding mutant body, that described toxin shows improvement or reduce kill insect active, or can encoding amino acid sequence, that described aminoacid sequence is given the polypeptide improvement that comprises it or reduce kill insect active.As used herein, the contextual term of albumen, polypeptide or aminoacid sequence " mutant " or " sudden change " refer to the sequence through suddenling change or comprising one or more amino-acid residues through transformation, and described amino-acid residue is not present in corresponding wild-type sequence.Such sudden change or one or more interpolations, disappearance or the replacement or the replacement institute that change by amino-acid residue form.That mutant polypeptide shows improvement or reduce kill insect active, or represented amino acid sequence, described aminoacid sequence is given the insect active that kills of the polypeptide improvement that comprises it.Thereby, one of term " mutant " or " sudden change " phalangeal process variant nucleotide sequence and coded amino acid or both.Mutant can use separately or with other mutant or with other mutant of embodiment consistently arbitrary combination use." mutant polypeptide " can show the reduction of killing insect active on the contrary.While will the sudden change over being added into specific nucleic acid or albumen, described sudden change can be added simultaneously or sequentially; If order is added, sudden change can be added with any suitable order.

As used herein, what term " improvement kill insect active " or " insecticidal activity of improvement " related to embodiment kills insect polypeptide, described polypeptide has the insect active that kills of enhancing with respect to its corresponding wild-type protein, and/or relate to the insect of wide spectrum is more effectively killed to insect polypeptide, and/or relate to and there is the specific insect polypeptide that kills to not being subject to the insect of wild-type protein toxic effect.The discovery of insecticidal activity improvement or that strengthen need to prove the insecticidal activity of killing insect polypeptide with respect to wild-type, and the insecticidal activity of insect target is improved at least 10%, or insecticidal activity improves at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 100%, 150%, 200% or 300% or more, the definite of described activity is for same insect.

For example, the desinsection of improvement be provided or kill insect active, wherein with respect to the scope of the insect that affected by wild-type Bt toxalbumin, being subject to the scope of the insect that this polypeptide affects wider or narrower.When needs are multi-functional, may need wider range of influence, and when for example useful insect is likely subject in addition the use of this toxin or has affect, may need narrower range of influence.Although embodiment is not subject to the restriction of any special mechanism of action, also can provide by changing one or more characteristics of polypeptide the insecticidal activity of improvement; For example, can increase stability or the life-span of polypeptide in insect intestines, described increase is with respect to stability or the life-span of corresponding wild-type protein.

Term used herein " toxin " refers to show insecticidal activity or kills insect active or the polypeptide that kills insect active of the insecticidal activity of improvement or improvement." Bt " or " bacillus thuringiensis " toxin is intended to be included in the Cry toxin of the wide variety of finding in the various bacterial strains of Bt, and it comprises such as for example Cry1s, the toxalbumin of Cry2s or Cry3s.

Term " proteolysis site " or " cleavage site " refer to aminoacid sequence, and it gives a proteinoid enzyme or special proteolytic enzyme by susceptibility, so that the polypeptide that comprises described aminoacid sequence is digested by such proteolytic enzyme or special proteolytic enzyme.It is said, proteolysis site is to identifying one or more proteolytic enzyme " sensitivity " in this site.This area is understood, and digestive efficiency is different, and the reduction of digestive efficiency can cause the increase of this polypeptide in insect intestines internal stability or life-span.Thereby susceptibility can be given more than a kind of proteolytic enzyme or a more than proteinoid enzyme in proteolysis site, but various proteolytic enzyme can be different to the digestive efficiency in this site.Proteolysis site comprises, for example, and trypsinase site, Chymotrypsin site and elastoser site.

For example, research has shown that lepidopterous insect ereptase comprises trypsinase, Chymotrypsin and elastoser.Referring to, for example, Lenz et al. (1991) Arch.Insect Biochem.Physiol.16:201-212; With Hedegus et al. (2003) Arch.Insect Biochem.Physiol.53:30-47.For example, in the middle intestines of Helicoverpa armigera larva, found that approximately 18 kinds of different trypsin are referring to Gatehouse et al. (1997) Insect Biochem. Mol.Biol.27:929-944).Studied the proteolysis substrate sites of these proteolytic enzyme institute preferences.Referring to, Peterson et al. (1995) Insect Biochem.Mol.Biol.25:765-774 for example.

The character through engineering approaches toxin of having made great efforts to understand the mechanism of action of Bt toxalbumin and having utilized improvement.Shown that insect ereptase can affect the impact of Bt Cry albumen on this insect.Some proteolytic enzyme, by Cry albumen is machined to toxic forms or " toxin " from " toxogen (protoxin) " form, activate Cry albumen.Referring to Oppert (1999) Arch.Insect Biochem.Phys.42:1-12; With Carroll et al. (1997) J.Invertebrate Pathology 70:41-49.The activation of this toxalbumin can comprise from described albumen and remove N-terminal and C-terminal peptide, and can comprise and from inside, cut this albumen.Other proteolytic enzyme described Cry albumen of can degrading.Referring to Oppert, as front.

The sequence units (block) that has relatively disclosed 5 high conservatives of the aminoacid sequence of not homospecific Cry toxin.Described toxin structurally comprises 3 different structural domains, from N-terminal to C-terminal, is: bunch (being called " structural domain 1 "), 3 the antiparallel β lamellas (being called " structural domain 2 ") that relate to Cell binding and the β sandwich (being called " structural domain 3 ") that relate to 7 α spirals of hole formation.The position of known these structural domains of those skilled in the art and character.Referring to, for example, Li et al. (1991) Nature, 305:815-821 and Morse et al. (2001) Structure, 9:409-417.Relate to ad hoc structure territory, for example, while relating to structural domain 1, the definite terminal that should understand about the structural domain of particular sequence is not crucial, as long as the sequence providing owing at least some functions in this ad hoc structure territory is provided for this sequence or its part.Thereby, for example, when mentioning " structural domain 1 ", be intended to refer to particular sequence comprise 7 α spirals bunch, but sequence used or be not critical about the definite terminal of the sequence of this bunch.Those skilled in the art are familiar with determining and the evaluation to this class function of this class terminal.

In order to attempt characterizing better and improve Bt toxalbumin, studied Bt bacterial isolates.The crystal prepared product that discovery is prepared from Bt strain culture has the insecticidal activity (seeing embodiment 1) of anti-west corn rootworm.Attempt identifying the nucleotide sequence from the coding crystallin of selected bacterial strain, wild-type (naturally occurring) nucleic acid from the separated embodiment of these bacterial isolateses, is cloned into expression vector and is transformed into intestinal bacteria.Rely on the characteristic of given prepared product, recognize that the proof of insecticidal activity needs trypsinase pre-treatment to activate this insecticidal proteins sometimes.For example, thereby the activation that should understand some insecticidal proteins needs protease digestion (, by trypsinase, Chymotrypsin etc.), and other albumen is for example, without under activation being bioactive (, pesticidal).

Can be by the U. S. application the 10/606th of submitting on June 25th, 2003, the 10/746th of No. 320 and submission on December 24th, 2003, the method described in No. 914 is transformed described molecule.In addition, can be by nucleotide sequence through engineering approaches, the polypeptide with encoded packets containing other sudden change, described sudden change is given with respect to the insecticidal activity improvement of the insecticidal activity of naturally occurring polypeptide or that change.The nucleotide sequence of the nucleic acid of this project comprises the sudden change of not finding in wild-type sequence.

Conventionally by the method comprising the following steps, prepare the mutant polypeptide of embodiment: the nucleotide sequence that obtains coding Cry family polypeptides; Analyze the structure of described polypeptide, the mutagenesis of the latent gene sequence in specific to identify " target " site, this consideration based on the function of proposing in detoxifying function pattern to described target structure territory; One or more sudden changes are introduced to nucleotide sequence to produce the required change to one or more amino-acid residues of coded peptide sequence; And the insecticidal activity of analyzing the polypeptide that produces.

Bt kills many in insect toxins and is correlated with, and the similarity degree of its aminoacid sequence and tertiary structure is different, and the method for known acquisition Bt toxalbumin crystalline structure.From document, can obtain the high resolving power crystallographic structural analysis of exemplary Cry3A and Cry3B polypeptide.The Cry3A gene structure being resolved to (Li et al. (1991) Nature 353:815-821) provide the profound understanding to relation between this toxin structure and function.The published structural analysis of Bt toxin and the function relevant to ad hoc structure, motif etc. of reporting are considered in combination, illustrate that the discontinuous step of the specific region of this toxin and the specific function of this albumen and specific function pattern is relevant.For example, by separation, the many toxin from Bt are described as comprising three structural domains conventionally: relate to 7 helical bundles, the 3 laminated structure territories that relate to receptors bind and β sandwich motif (Li et al. (1991) Nature 353:815-821) that hole forms.

As United States Patent (USP) the 7th, the unsettled U. S. application the 10/746th that 105, No. 332 and on December 24th, 2003 submit to, No. 914 institute reports, can, by the region between the α spiral 3 and 4 in this toxin structure territory 1 as target, improve the toxicity of Cry albumen.This theory builds on about killing the knowledge hierarchy of insect toxins, comprising: 1) it is reported that the α spiral 4 and 5 in Cry3A toxin structure territory 1 inserts lipid bilayer Gazit etal. (1998) the Proc.Natl.Acad.Sci.USA 95:12289-12294 of the cell of liner susceptible insect midgut); 2) contriver understands the position of wild-type protein aminoacid sequence endotrypsin and Chymotrypsin cleavage site; 3) observe after the Activated in Vitro by trypsinase or Chymotrypsin processing, wild-type protein has more activity to some insect; And 4) report has caused the reduction to insect toxicity from 3 ' end peptinotoxin.

Can prepare a series of sudden changes, and be placed in diversity of settings sequence, thereby preparation has the new polypeptide of insecticidal activity enhancing or that change.Referring to, for example, the U. S. application the 10/606th of nowadays submitting in resigned, on June 25th, 2003, No. 320 and submit on December 24th, 2003 the 10/746th, No. 914.These mutant include but not limited to: at least one protease-sensitive site (for example trypsinase cutting site) is added in the region between the spiral 3 and 4 at structural domain 1 in addition; With the protease-sensitive site of difference, replace the site of the original protein enzyme sensitivity of wild-type sequence; At specific position, add the responsive site of polyprotein enzyme; Near one or more protease-sensitive sites, add amino-acid residue, thereby change the also folding of described polypeptide thereby strengthen the polypeptide digestion in these one or more protease-sensitive site; To protect this polypeptide to avoid, make the degradation property that toxicity reduces digest (for example, prepare a series of sudden changes, wherein with α-amino-isovaleric acid, replace wild-type amino acid to protect described polypeptide to avoid digestion) with adding to suddenly change.Can use sudden change separately or with arbitrary combination, thereby the polypeptide of embodiment is provided.

Embodiment provides the sequence that comprises various sudden changes by this way, described sudden change such as, for example comprise the sudden change in the responsive site of other or selectable proteolytic enzyme between the α spiral 3 and 4 in coded polypeptide structure territory 1.Sudden change in addition or the responsive site of selectable proteolytic enzyme can be to several proteolytic enzyme such as serine protease or responsive such as the enzyme of elastoser, and described serine protease comprises trypsinase and Chymotrypsin.Thereby sudden change in addition or the responsive site of selectable proteolytic enzyme can easily be identified and/or cut by protease through design so that this site, described proteolytic enzyme is such as mammalian protease or insect protein enzyme.Protease-sensitive site can also be designed to be cut by known enzyme or the certain enzyme that results from the particular types of organism, described enzyme such as, the Chymotrypsin (Lenz et al. (1991) Arch.Insect Biochem.Physiol.16:201-212) that for example bollworm Heliothis zea produces.Sudden change can also be given the resistance to proteolytic digestion, for example the resistance in the digestion of the C-terminal of described peptide to Chymotrypsin.

There is the responsive site of other and/or selectable proteolytic enzyme in the aminoacid sequence of coded polypeptide, this can improve insecticidal activity and/or the desinsection specificity of polypeptide of the nucleic acid encoding of embodiment.Therefore, the nucleotide sequence of embodiment can reorganized through engineering approaches or is operated to produce and have insecticidal activity and/or specific polypeptide improvement or that change, described improvement or change with respect to not modified wild-type toxalbumin.In addition, sudden change disclosed herein can be placed in other nucleotide sequence or with other nucleotide sequence coupling, thereby the character of improvement is provided.For example, the insect Chymotrypsin easily responsive site of proteolytic enzyme of cutting can be placed in Cry background sequence, so that the toxicity for the improvement of this sequence to be provided, the Chymotrypsin that described insect Chymotrypsin is for example found in lace mythimna separata (bertha armyworm) or bollworm (Hegedus et al. (2003) Arch.Insect Biochem.Physiol.53:30-47; With Lenz et al. (1991) Arch.Insect Biochem.Physiol.16:201-212).Embodiment provides the toxic polypeptide with improved properties by this way.

For example, the Cry nucleotide sequence of sudden change can comprise other mutant, and described mutant comprises the other codon of the aminoacid sequence of the second trypsinase sensitivity (except naturally occurring trypsinase site) being introduced to coded polypeptide.The selectable interpolation mutant of embodiment comprises other codon, described codon is through designing so that this polypeptide is introduced at least one other different protease-sensitive site, for example,, near the site of the Chymotrypsin sensitivity of naturally occurring trypsinase site 5 ' or 3 '.Selectively, can prepare replacement mutant, wherein destroy at least one codon of the nucleic acid in the responsive site of the naturally occurring proteolytic enzyme of coding, and selectable codon is introduced to nucleotide sequence, thereby different (for example replacing) protease-sensitive sites is provided.Substitution body can also be added into Cry sequence, wherein destroy the naturally occurring trypsinase cutting site existing in coded polypeptide, and by Chymotrypsin or its position of elastoser cleavage site introducing.

Recognize any nucleotide sequence of the proteolysis site that can use proteins encoded hydrolysis site or infer (for example such as NGSR, RR or LKM sequence) aminoacid sequence, and for the codon of any site of these cleavage sites being introduced to variant polypeptide really personal part can be according to purposes and difference, described purposes i.e. expression in specified plant species.Also recognize that any sudden change in disclosed sudden change can be introduced into arbitrary polynucleotide sequence of embodiment, the codon that described sequence comprises the amino-acid residue that natural trypsinase cutting site is provided, described cleavage site is the target of modifying.Therefore, can modify the variant of total length toxin or its fragment, to comprise other or selectable cleavage site, and these embodiments to be intended to the scope of embodiment disclosed herein included.

Skilled person in the art will appreciate that any useful sudden change may be added to the sequence of embodiment as long as coded polypeptide retains insecticidal activity.Thereby, also can mutant nucleotide sequence, thus coded polypeptide has resistance to the proteolytic digestion of Chymotrypsin.Can add the recognition site that surpasses with any specific position that is combined in, and can add or remove many recognition sites from described toxin to described toxin.Thereby sudden change in addition can comprise 3,4 or more recognition site.By recognize can be in arbitrary suitable polynucleotide sequence through engineering approaches multimutation; Therefore, can modify full length sequence or its fragment, to comprise other or selectable cleavage site and proteolytic digestion is had to resistance.So, embodiment provides the Cry that comprises sudden change toxin, and described sudden change improves insecticidal activity, and embodiment also provides the composition of improvement and uses other Bt toxin to affect the method for insect.

Sudden change can protect this polypeptide to avoid proteasome degradation, and described protection is for example by remove the proteolysis site of inferring such as the serine protease site of inferring and elastin enzyme recognition site from different zones.Can remove or transform some or all that these infer site, so that the proteolysis of position, original site is lowered.Can be by comparing mutant polypeptide and wild-type toxin or by the different mutant toxin of comparing amino acid sequence, evaluating proteoclastic variation.Proteolysis site and the proteolysis site of inferring include but not limited to following sequence: RR, trypsinase cutting site; LKM, Chymotrypsin site; And NGSR, trypsinase site.As long as the insecticidal activity of described polypeptide is enhanced, can transform these sites by the amino-acid residue adding or lack arbitrary number and kind.Thereby, with respect to native sequences or background sequence, by the nucleotide sequence coded polypeptide that comprises sudden change, at least one amino acid change or interpolation will be comprised, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 35, 38, 40, 45, 47, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270 or 280 or more amino acid change or interpolation.As known in the art, can also be by brachymemma natural or full length sequence improve the insecticidal activity of polypeptide.

The composition of embodiment comprises nucleic acid and fragment and its variant, its coded insect-killing polypeptide.Especially, embodiment provides separated nucleic acid molecule, the nucleotide sequence that described nucleic acid molecule comprises the aminoacid sequence shown in coding SEQID NO:2, or the nucleotide sequence that comprises encode such amino acid sequences, the for example nucleotide sequence shown in SEQ ID NO:1, and fragment and its variant.

What receive publicity in addition is the optimization nucleotide sequence of the insecticidal proteins of coding embodiment.As used herein, phrase " optimization nucleotide sequence " refers to that described specific organism is for example plant in order to express the nucleic acid of optimizing in specific organism.Can use means known in the art for the preparation of the optimization nucleotide sequence of any object organism.Referring to, for example, the U. S. application the 10/606th of nowadays submitting in resigned, on June 25th, 2003, No. 320 and submit on December 24th, 2003 the 10/746th, No. 914, they have described the optimization nucleotide sequence of the disclosed insecticidal proteins of encoding.In the present embodiment, the preparation of described nucleotide sequence is by the aminoacid sequence of reverse translation albumen, and changes nucleotide sequence so that comprise corn preference codon but still the identical aminoacid sequence of encoding.The more details of the method are described in Murray et al. (1989) Nucleic Acids Res.17:477-498.Optimize nucleotide sequence and can be used for improving the expression of insecticidal proteins in plant, described plant is (Poaceae) monocotyledons, for example corn (maize or corn) plant of Gramineae (Gramineae) for example.

Embodiment also provides separated desinsection (for example killing insect) polypeptide, and its nucleic acid by the naturally occurring of embodiment or modification is coded.More specifically, embodiment provides the polypeptide that comprises aminoacid sequence shown in SEQID NO:2, and the polypeptide of nucleic acid encoding described herein, and nucleic acid described herein is the nucleic acid shown in SEQ ID NO:1 for example, and fragment and its variant.

In specific implementations, the insecticidal proteins of embodiment provides total length to kill insect polypeptide, total length is killed the fragment of insect polypeptide and the variant polypeptide being produced by mutant nucleic acid, and described mutant nucleic acid is introduced specific amino acid sequence through design the polypeptide of embodiment.In specific implementations, the sequence providing such as the cleavage site of the enzyme of proteolytic enzyme is provided the aminoacid sequence that is introduced into described polypeptide.

The insecticidal activity of Bt toxin known in the art is activated by the cutting to described peptide in insect intestines of various proteolytic enzyme conventionally.Because peptide in insect intestines not always entirely to imitate cutting, so the fragment of total length toxin can have the insecticidal activity of enhancing with respect to total length toxin self.Thereby, some in the polypeptide of embodiment comprise that total length kills the fragment of insect polypeptide, and some in polypeptide fragment, varient and sudden change by with respect to its derived from naturally occurring activity of killing insect polypeptide, the insecticidal activity with enhancing, if particularly the naturally occurring insect polypeptide that kills does not carry out Activated in Vitro with proteolytic enzyme before screening activity.Thereby the application comprises clipped form or the fragment of described sequence.

Sudden change can be inserted comprise described by arbitrary background sequence of brachymemma polypeptide, as long as described polypeptide retains insecticidal activity.Use the analysis of analysis known in the art or elsewhere description herein, those skilled in the art can easily compare the insecticidal activity of two or more albumen.Can be by expressing nucleic acid disclosed herein or producing by use standard molecular biological technique by the polypeptide of understanding embodiment.

Recognize described insecticidal proteins can be oligomerization and can molecular weight, residue number, component polypeptide, anti-specific insect is active and other characteristic on different.Yet, by methods described herein, can separatedly also characterize the activated albumen of various insects.The insecticidal proteins of embodiment can be used in combination with other Bt toxin or other insecticidal protein, thereby improves target insect scope.In addition, the insecticidal proteins of embodiment and other Bt toxin or of different nature other kill the given efficacy that being used in combination of insect principle has prevention and/or process insect-resistant.Other insecticide comprises proteinase inhibitor (Serine and halfcystine type), α-amylase and peroxidase.

Embodiment also comprises the aminoacid sequence of Nucleotide and coding thereof and the fragment of polypeptide and variant.As used herein, term " fragment " refers to a part for nucleotide sequence for polynucleotide or a part for the aminoacid sequence of polypeptide for embodiment.The fragment of nucleotide sequence can proteins encoded fragment, and described protein fragments retains the biologic activity of natural or corresponding full-length proteins, and thereby has an insecticidal activity.Thereby the polynucleotide of known embodiments and some in aminoacid sequence can correctly be called fragment and mutant.

Should understand if term " fragment " is when mentioning the nucleotide sequence of embodiment, this term also comprises the sequence that can be used as hybridization probe.This class nucleotide sequence is not conventionally encoded and is retained the Fragment Protein of biologic activity.Thereby the scope of the fragment of nucleotide sequence can be from least about 20 Nucleotide, approximately 50 Nucleotide, approximately 100 Nucleotide are to the full length nucleotide sequence of the albumen of coding embodiment.

The nucleotide sequence fragment of the embodiment of the insecticidal proteins biologic activity part of coding embodiment will encode at least 15,25,30,50,100,200,300,400,500,600 or 700 continuous amino acid, or coding reaches the amino acid (for example 719 of SEQ ID NO:2 amino acid) of all amts existing in the insecticidal peptide of embodiment.Thereby, should understand embodiment and also comprise polypeptide, described polypeptide be embodiment exemplary insecticidal proteins fragment and have to the youthful and the elderly 15,25,30,50,100,200,300, the amino acid (for example 719 of SEQ ID NO:2 amino acid) of all amts existing in the insecticidal peptide of 400,500,600 or 700 continuous amino acids or nearly embodiment.The nucleotide sequence fragment that can be used as the embodiment of hybridization probe or PCR primer does not need the biologic activity part of encoding insecticidal proteins conventionally.Thereby, the biologic activity part that the nucleic acid fragment of embodiment can encoding insecticidal proteins, or it can be can be as using the hybridization probe of method disclosed herein or the fragment of PCR primer.The preparation of the biologic activity part of insecticidal proteins can be by a kind of part in the nucleotide sequence of separated embodiment, express the coded part (for example passing through in-vitro recombination expression) of insecticidal proteins, and analyze the activity of the coded part of described insecticidal proteins.

Nucleic acid as embodiment nucleotide sequence fragment comprises at least 16,20,50,75,100,150,200,250,300,350,400,450,500,600,700,800,1000,1200,1300 or 1400 Nucleotide, or reach the Nucleotide (for example 1406 of SEQ ID NO:1 Nucleotide) of existing quantity in nucleotide sequence disclosed herein.Specific implementations relates to for example, fragment derived from first nucleic acid of (produce from) embodiment, and wherein said fragment coding be take the toxin of the brachymemma that insecticidal activity is feature.The polypeptide of the brachymemma that the polynucleotide passage of embodiment is coded is characterised in that the insecticidal activity of equivalence or improvement, described equivalence or improvement be with respect to this fragment derived from the activity of the coded corresponding full-length polypeptide of the first nucleic acid.This nucleic acid fragment of related is embodiment can be at 3 ' end of natural or corresponding complete encoding sequence by brachymemma.Nucleic acid fragment can also be held all by brachymemma at 5 ' and 3 ' of natural or corresponding complete encoding sequence.

Term used herein " variant " refers to substantially similar sequence.For nucleotide sequence, conservative variant comprises due to encode those sequences of a kind of aminoacid sequence in the insecticidal peptide of embodiment of genetic codon degeneracy.Such as these naturally occurring allele variant, can use known Protocols in Molecular Biology to identify, described technology such as, listed polymerase chain reaction (PCR) and hybridization technique herein for example.

Variant nucleotide sequence also comprises synthetic derivative nucleotide sequence, and those of the insecticidal proteins of the embodiment of still encoding producing such as using for example site-directed mutagenesis, such as mutant toxin.In general, the variant of the specific nucleotide sequence of embodiment will have at least about 70%, 75% with this specific nucleotide sequence, and 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity, conformingly determine it is by the sequence alignment program described in this paper elsewhere, described program is used default parameters.The nucleotide sequence variant of embodiment and the difference of this sequence may be as few as 1-15 Nucleotide, few for example, to 1-10 (6-10), few to 5, few to 4,3, and 2 or 1 Nucleotide even.

The variant of the specific nucleotide sequence of Evaluation operation mode (being Exemplary core nucleotide sequence) can also be by comparing the sequence identity per-cent of the coded polypeptide of variant nucleotide sequence and the coded polypeptide of this reference nucleotide sequence.Thereby for example, disclosing encodes has the separated nucleic acid of the polypeptide of given sequence consistence per-cent with the polypeptide of SEQ ID NO:2.Can use herein sequence alignment program described in elsewhere to calculate the sequence identity per-cent between any two polypeptide, described program is used default parameters.If by comparing any given sequence identity per-cent that two coded polypeptide of polynucleotide are enjoyed of embodiment, it is described any given right to evaluate, the sequence identity per-cent between these two coded polypeptide is at least about 40%, 45% so, 50%, 55%, 60%, 65%, 70%, generally at least about 75%, 80%, 85%, at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or at least about 98%, 99% or higher sequence identity.

As used herein, term " misfolded proteins " comprises the polypeptide derived from native protein, and described deriving is by lacking one or more amino acid of (so-called brachymemma) native protein N-terminal and/or C-terminal or N-terminal and/or the C-terminal to described native protein by one or more aminoacid addition; In the one or more sites of native protein disappearance or add one or more amino acid; Or the one or more sites at native protein replace one or more amino acid.Thereby term " misfolded proteins " comprises the bioactive fragment of native protein, it comprises and retains a day hot biological activity of albumen, has the continuous amino acid residue of enough numbers of insecticidal activity.The relative native protein of such insecticidal activity can be different or improvement, or it can be constant, as long as retained insecticidal activity.

The misfolded proteins that embodiment comprises has biologic activity, and they continue to have the required biologic activity of native protein, and described activity is insecticidal activity as herein described.Described variant can derive from gene pleiomorphism for example or the mankind's operation.The biologic activity variant of the natural insecticidal proteins of embodiment will have at least about 60%, 65% with native protein aminoacid sequence, and 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity, conformingly determine it is the sequence alignment program of describing by this paper elsewhere, described program is used default parameters.The biologic activity variant of the albumen of embodiment and the difference of this albumen may be as few as 1-15 amino-acid residue, few for example, to 1-10 (6-10), few to 5, few to 4,3, and 2 or 1 amino-acid residue even.

Embodiment also comprises the microorganism of conversion, the carrier that described conversion is used the nucleic acid of at least one embodiment, the expression cassette that comprises described nucleic acid or comprised described expression cassette.In some embodiments, described microorganism is the microorganism that relies on plant propagation.Embodiments of the present invention relate to the insecticidal proteins of encapsulation, and described albumen comprises the microorganism of the conversion of the insecticidal proteins that can express at least one embodiment.

Embodiment provides insect-killing composition, comprises the microorganism of the conversion of embodiment.In this embodiment, the microorganism of described conversion is present in described insect-killing composition jointly with insecticidal effective dose and suitable carrier conventionally.Embodiment also comprises insect-killing composition, described composition comprises the separated albumen of the embodiment that kills insect significant quantity, and suitable carrier, the albumen Individual existence of described separation or with the combination of the organism of conversion of embodiment and/or the insecticidal proteins of the encapsulation of embodiment.

Embodiment also provide use the insecticidal proteins of embodiment and at least one other or the combination of " second " insecticidal proteins, increase the method for insect target scope.Any insecticidal proteins known in the art can be used for to the method for embodiment.Described insecticidal proteins includes but not limited to Bt toxin, proteinase inhibitor, α-amylase and peroxidase.

Embodiment also comprises plant or the transgenic plant of conversion, the nucleotide sequence that it comprises at least one embodiment.In some embodiments, use constructs stably to build plant, the nucleotide sequence that described construct comprises at least one embodiment being operably connected with the promotor that drives expression in vegetable cell.As used herein, term " plant of conversion " and " transgenic plant " represent the plant that comprises heterologous polynucleotide in genome.In general, described heterologous polynucleotide is stably integrated in the genome of the plant of transgenosis or conversion, so that described polynucleotide are passed to offspring.Described heterologous polynucleotide can be integrated into genome individually or as the part of recombinant expression cassettes.

Should understand as used herein, term " genetically modified " comprises genotype reformed any cell, clone, callus, tissue, plant part or plant by the existence of heterologous nucleic acids, comprise initial those transgenosiss that so change, and by sexual hybridization or vegetative propagation from those of described initial transgenosis generation.As used herein, term " genetically modified " does not comprise by conventional plant breeding method or the change to genome (karyomit(e) or karyomit(e) are outer) by natural event, described natural event such as random cross fertilization, non-recombinant virus infection, the conversion of non-recombinant bacteria, non-restructuring swivel base or spontaneous mutation.

As used herein, term " plant " comprises whole plant, plant organ (for example leaf, stem, root etc.), seed, vegetable cell and filial generation thereof.The part of transgenic plant belongs to the scope of embodiment, comprise, for example, come from transgenic plant or its filial generation and thereby vegetable cell, protoplastis, tissue, callus, embryo and the flower, stem, fruit, leaf and the root that by transgenic cell, are formed at least partly, described transgenic plant first transform with the DNA molecular of embodiment.

As used herein, term plant comprises vegetable cell, plant protoplast, Plant cell and tissue culture thing, plant callus, agglomerate and the vegetable cell of the plant that can regenerate, and it is the part of complete plant or plant, such as embryo, pollen, ovule, seed, leaf, flower, branch, fruit, kernel, fringe, cob, shell, stalk, root, the tip of a root, flower pesticide etc.Can be conventionally equally wide in range with the higher plant kind that is suitable for transformation technology for the floristics of the method for embodiment, comprise monocotyledons and dicotyledons.Described plant comprises for example potato (Solanum tuberosum) and corn (Zea mays).

Although embodiment does not rely on particular biological mechanism, do not increase the resistance of plant to plant insect, the expression that the nucleotides sequence of embodiment is listed in plant can cause producing the insecticidal proteins of embodiment and improve the resistance of described plant to plant insect.The plant of embodiment can be for affecting the method for insect pest in agricultural.Some embodiment provides the crop plants of conversion, and such as for example maize plant, it can be for affecting the method for the insect pest of this plant, and described insect pest is such as for example west corn rootworm.

" tested plant or vegetable cell " is plant or the vegetable cell that the genetic modification such as transforming of goal gene is entered into force, or passes plant or cell and the plant that comprises described transformation or the vegetable cell from so transformation." contrast " or " control plant " or " control plant cell " is provided for measuring the reference point of tested plant or vegetable cell phenotypic alternation.

Control plant or vegetable cell can comprise, for example: (a) wild-type plant or cell, there is plant or the cell of homologous genes type with genetic modification parent material, described genetic modification produces tested plant or cell; (b) there is homologous genes type with described parent material but plant or the vegetable cell of having used empty construct (using the construct of object proterties endlessly being known to effect, such as the construct that comprises marker gene) to transform; (c) be plant or the vegetable cell of the non-transformed segregant of tested plant or vegetable cell; (d) consistent but be not exposed to plant or the vegetable cell of conditioned disjunction stimulator that can induction destination gene expression in heredity with described tested plant or vegetable cell; Or (e) tested plant or vegetable cell self, under its condition not being expressed in goal gene.

Those skilled in the art can easily admit, progress such as the biology field of site-specific mutagenesis and random mutagenesis, polymerase chain reaction method and protein engineering technology provides proper implements and operation steps widely, for the aminoacid sequence of interested albumen and potential gene order in transformation or through engineering approaches agricultural.

Thereby the albumen of embodiment can be transformed in every way, comprises aminoacid replacement, disappearance, brachymemma and insertion.Method for described operation is well known in the art.For example, can introduce by suddenling change the aminoacid sequence variant that nucleic acid (for example DNA molecular) is prepared insecticidal proteins.Method for mutagenesis and nucleic acid change is well known in the art.For example, can use oligonucleotide mediated site-directed induced-mutation technique, introduce the change through design.Referring to, Kunkel (1985) Proc.Natl.Acad.Sci.USA 82:488-492 for example; Kunkel et al. (1987) Methods in Enzymol.154:367-382; United States Patent (USP) the 4th, 873, No. 192; Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (Protocols in Molecular Biology) (MacMillan Publishing Company, New York), and the document of quoting herein.

Can modify the nucleotide sequence of the sudden change of embodiment, thereby change approximately 1,2,3,4,5,6,8,10,12 or the more amino acid that is present in coded polypeptide primary sequence.Selectively, can introduce the more change to native sequences, consequently coded albumen can have at least about 1% or 2%, or approximately 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, or approximately 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, 21%, 22%, 23%, 24%, or 25%, 30%, 35%, or 40% or the codon that more changes with respect to corresponding wild-type protein or modify in addition.In the same way, coded albumen can have at least about 1% or 2%, or approximately 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, or approximately 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, 21%, 22%, 23%, 24%, or 25%, 30%, 35%, or 40% or the codon that more adds with respect to corresponding wild-type protein.The nucleotide sequence that should be understood that the sudden change of embodiment is intended to comprise peptide biological function, that be equal to insecticidal activity, and described insecticidal activity is such as the insecticidal activity of improvement, and its antifeedant character by anti-west corn rootworm larva is determined.The appearance of described sequence can be because of codon redundancy and functional being equal to, and known its exists natively in the albumen of nucleotide sequence and coding like this.

One skilled in the art will realize that aminoacid addition and/or replace conventionally based on the substituent relative similarity of amino acid side chain, for example, described substituent hydrophobicity, electric charge, size etc.The sour substituted radical of exemplary amino with various aforementioned considered character is conventionally known to one of skill in the art, and comprises arginine and Methionin; L-glutamic acid and aspartic acid; Serine and Threonine; Glutamine and l-asparagine; And α-amino-isovaleric acid, leucine and Isoleucine.

Can be at Dayhoff et al. (1978) Atlas of Protein Sequence and Structure (protein sequence and structure atlas) (Natl.Biomed.Res.Found. about not affecting the guide of the suitable aminoacid replacement of target protein biologic activity, Washington, D.C) find in the model of (be incorporated to by reference herein).Can carry out another amino acid whose conservative property replacement of making to have similar quality such as an amino acid is changed.

The gene of embodiment and nucleotide sequence thereby comprise naturally occurring sequence and mutant forms.Similarly, the albumen of embodiment comprises naturally occurring albumen and variant (for example polypeptide of brachymemma) and its modified (for example mutant) form.Described variant has required insecticidal activity by continuing.Obviously, must not make described sequence not meet frame the sudden change producing in the nucleotide sequence at the described variant of coding, and described sudden change can not produce the complementary district that can produce mRNA secondary structure conventionally.Referring to, the open text the 75th of european patent application, No. 444.

Disappearance, insertion and the replacement of included protein sequence herein should not produce violent change to the character of described albumen.Yet, before replacing, lacking or inserting, while being difficult to predict its precise effects, it will be appreciated by those skilled in the art that described effect will be analyzed and be evaluated by the routine screening of analyzing such as insect feed.Referring to, for example, be incorporated to by reference Marrone et al. (1985) J.Econ.Entomol.78:290-293 and Czapla and Lang (1990) J.Econ.Entomol.83:2480-2485 herein.

Variant nucleotide sequence and albumen also comprise derived from the mutagenesis of resetting such as DNA and sequence and the albumen of recombination method.Utilize the method, can operate one or more different encoding sequences, thereby produce the new insecticidal proteins with required character.So, can produce recombination of polynucleotide library from correlated series polynucleotide group, described correlated series polynucleotide comprise the sequence area with basic sequence identity, and can be in vivo or external by homologous recombination.For example, use the method, can be between the nucleotide sequence of embodiment and the corresponding part of other known Cry nucleotide sequence any fragment of recombinant full-lenght encoding sequence, the sequence motifs of coding object structural domain or the nucleotide sequence of embodiment, thereby obtain new gene, described new genes encoding has the albumen of the object character of improvement.

The character receiving publicity includes but not limited to the insecticidal activity, protein stability of per unit insecticidal proteins, to non-target species, particularly to the mankind, domestic animal and the plant of insecticidal peptide and the toxicity of microorganism of expressing embodiment.Embodiment is not subject to the restriction of specific re-arrangement strategy, relates to nucleotide sequence or its part of at least one embodiment except described re-arrangement strategy.Rearrangement can only relate to nucleotide sequence disclosed herein, or can relate in addition rearrangement other nucleotide sequence known in the art.DNA re-arrangement strategy is known in the art.Referring to, for example, Stemmer (1994) Proc.Natl.Acad.Sci.USA 91:10747-10751; Stemmer (1994) Nature 370:389-391; Crameri et al. (1997) Nature Biotech.15:436-438; Moore et al. (1997) J.Mol.Biol.272:336-347; Zhang et al. (1997) Proc.Natl.Acad.Sci.USA 94:4504-4509; Crameri et al. (1998) Nature 391:288-291; And United States Patent (USP) the 5th, 605, No. 793 and the 5th, 837, No. 458.

The nucleotide sequence of embodiment can also be used for from the separated corresponding sequence of other organism, described other organism is other bacterium particularly, more in particular other Bacillus strain.So, can use such as the method for PCR, hybridization etc. and identify these sequences, the sequence homology of described evaluation based on itself and sequence described herein.Sequence identity based on itself and complete sequence described herein or its fragment and the sequence selected are also for embodiment is included.These sequences comprise the sequence as disclosed sequence homology thing.Term " homologue " refers to form derived from common ancestor's gene and because of species the gene find in different plant species.For the gene of finding, if its nucleotide sequence and/or its coded protein sequence are as his place of this paper defines and enjoys basic sequence identity, be considered to homologue in different plant species.The function of homologue high conservative usually between species.

In PCR method, can be designed for the Oligonucleolide primers of PCR reaction, with from extracting from the cDNA of any object organism or the genomic dna corresponding DNA sequence dna that increases.Design PCR primer and PCR clone's method is generally known in the art, and be disclosed in Sambrook et al. (1989) Molecular Cloning:A Laboratory Manual (molecular cloning: laboratory manual) (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York), below claim " Sambrook ".Separately referring to Innis et al., eds. (1990) PCR Protocols:A Guide to Methods and Applications (PCR operation steps: method and application guide) (Academic Press, New York); Lnnis and Gelfand, eds. (1995) PCR Strategies (PCR strategy) (Academic Press, New York); And Innis and Gelfand, eds. (1999) PCR Methods Manual (PCR method handbook) (Academic Press, New York).The currently known methods of PCR includes but not limited to following method, and described method is used paired primer, nested primer, monospecific primer, degenerated primer, gene-specific primer, carrier specificity primer, part mispairing primer etc.

In hybridization technique, by all or part of probe of hybridizing with other corresponding nucleotide sequence selective that is used as of known nucleotide sequence, described other corresponding nucleotide sequence is present in from the fragment of cloned genomic dna of selected organism or cDNA segment group (being genomic library or cDNA library).Described hybridization probe can be genomic DNA fragment, cDNA fragment, RNA fragment or other oligonucleotide, and can use such as ³²p can detection moiety or other can detect mark and carry out mark.Thereby, for example, can prepare hybridization probe by the synthetic oligonucleotide based on embodiment sequence by mark.The method of preparing hybridization probe and construction cDNA and genomic library is generally known in the art, and is disclosed in Sambrook.

For example, can by complete sequence disclosed herein or its one or more parts as can with the probe of corresponding sequence and messenger RNA(mRNA) specific hybrid.For completing under various conditions specific hybrid, described probe comprises and is unique to embodiment sequence and the common long sequence at least about 10 or 20 Nucleotide.Can use described probe, with by PCR from the selected organism corresponding Cry sequence that increases.Can use this technology, with from separated other encoding sequence of required organism, maybe this technology is used as to diagnositc analysis, to determine whether organism exists encoding sequence.Hybridization technique comprises DNA library (plaque or the bacterium colony that screening by hybridization is inoculated; Referring to, for example, Sambrook).

Can under stringent condition, carry out the hybridization of described sequence.As used herein, term " stringent condition " or " tight hybridization conditions " are expressed as follows condition, under this condition, with respect to other sequence hybridization, probe will for example, with detectable (, at least 2 of background times, 5 times or 10 times) more and its target sequence hybridization.Stringent condition is sequence dependent and different in varying environment.By control, hybridize stringency and/or control cleaning condition, can identify the target sequence (homologous probe method) with described probe 100% complementation.Selectively, can regulate stringent condition, to allow some sequence mispairing, to detect lower similarity (allos probe method).Conventionally, probe length is less than approximately 1000 or 500 Nucleotide.

Conventionally, stringent condition is following condition, in this condition, salt concn is pH 7.0 to 8.3 times, be less than about 1.5M Na ion, common about 0.01M to 1.0M Na ionic concn (or other salt), and temperature be for short probe (for example 10 to 50 Nucleotide) at least about 30 ℃, and for long probe (being for example greater than 50 Nucleotide) at least about 60 ℃.Can also such as the destabilizing agent of methane amide, realize stringent condition by adding.Exemplary low stringent condition comprises methane amide damping fluid, 1M NaCl, 1%SDS (sodium lauryl sulphate) hybridization of using 30% to 35% at 37 ℃, at 50 ℃ to 55 ℃ 1 * to cleaning in 2 * SSC (20 * SSC=3.0M NaCl/0.3M trisodium citrate).Exemplary moderate stringent condition comprises at 37 ℃ and hybridizing in 40% to 45% methane amide, 1.0M NaCl, 1%SDS, at 55 ℃ to 60 ℃ 0.5 * to 1 * SSC, clean.Exemplary high stringent condition comprises at 37 ℃ and hybridizing in 50% methane amide, 1M NaCl, 1%SDS, finally cleans at least about 20 minutes at 60 ℃ to 65 ℃ in 0.1 * SSC.Optionally, cleaning buffer solution can comprise approximately 0.1% to about 1%SDS.The hybridization time length is less than approximately 24 hours conventionally, is generally approximately 4 hours to approximately 12 hours.

Specificity relies on the cleaning after hybridization conventionally, and key factor is ionic strength and the temperature of last cleaning solution.The T of DNA-DNA heterozygote _m(thermal melting point) can be similar to the formula from Meinkoth and Wahl (1984) Anal.Biochem.138:267-284: T _m=81.5 ℃+16.6 (%GC)-0.61, (log M)+0.41 (% methane amide)-500/L; Wherein M is the molconcentration of monovalent cation, and %GC is the percentage ratio of guanosine and cytidylic acid(CMP) in DNA, and " % methane amide " is the methane amide percentage ratio of hybridization solution, and L is the base pair length of heterozygote.T _mthe temperature of (under definite ionic strength and pH) complementary target sequence of 50% during with the probe hybridization mating completely.Conventionally cleaning is at least proceeded to and reaches balance, and reach low hybrid context level, such as carrying out 2 hours, 1 hour or 30 minutes.

Every 1% mispairing correspondence makes T _mreduce approximately 1 ℃; Thereby, can regulate T _m, hybridization and/or cleaning condition, thereby with required conforming sequence hybridization.For example, if need>=90% conforming sequence, can be by T _mreduce by 10 ℃.Conventionally, stringent condition is chosen as than the T of the distinguished sequence under definite ionic strength and pH and complementary sequence thereof _mlow approximately 5 ℃.Yet, under very tight condition, can be than described T _mat low 1 ℃, 2 ℃, 3 ℃ or 4 ℃, hybridize and/or clean; Under moderate stringent condition, can be than described T _mat low 6 ℃, 7 ℃, 8 ℃, 9 ℃ or 10 ℃, hybridize and/or clean; Under low stringent condition, can be than described T _mat low 11 ℃, 12 ℃, 13 ℃, 14 ℃, 15 ℃ or 20 ℃, hybridize and/or clean.

Use described formula, hybridization and cleaning combination, and required T _m, skilled person in the art will appreciate that the variation of having described in fact hybridization and/or cleaning solution stringency.If the mispairing of required degree causes the T lower than 45 ℃ (aqueous solution) or 32 ℃ (formamide solns) _m, can improve so SSC concentration so that can use higher temperature.To the general guide of nucleic acid hybridization referring to Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes (hybridization of biochemistry and molecular biology experiment technology-nucleic acid probe), Part I, Chapter 2 (Elsevier, New York); And Ausubel et al., eds. (1995) Current Protocols in Molecular Biology (the up-to-date experiment guide of molecular biology), Chapter 2 (Greene Publishing and Wiley-lnterscience, New York).Separately see Sambrook.Thereby embodiment comprises separated sequence, the Cry albumen of the sequence encoding embodiment of described separation and under stringent condition with Cry sequence disclosed herein or with the hybridization of its fragment.

With the sequence relation between two or more nucleic acid of following term description or polynucleotide: (a) " reference sequences ", (b) " comparison window (comparison window) ", (c) " sequence identity ", (d) " sequence identity per-cent " and (e) " basically identical property ".

(a) as used herein, " reference sequences " is the definite sequence as sequence comparison basis.Reference sequences can be subset or the integral body of specified sequence; For example, as the fragment of full-length cDNA or gene order, or complete cDNA or gene order.

(b) as used herein, " comparison window " relates to the fragment of the continuous and appointment of polynucleotide sequence, wherein the polynucleotide sequence of comparison window can comprise than reference sequences interpolation or the disappearance (being interval) of (it does not comprise interpolation or disappearance), so that two sequences are carried out to optimum ratio pair.Conventionally, comparison window is grown to few 20 continuous nucleotides, and optionally can grow 30,40,50,100 or longer.It will be appreciated by those skilled in the art that for avoid by polynucleotide sequence comprise interval that cause with high similarity reference sequences, conventionally introduce interval point penalty, and it deducted number from coupling.

Comparison method for comparative sequences is known in the art.Thereby, can use mathematical algorithm to complete determining sequence identity per-cent between any two sequences.The limiting examples of described mathematical algorithm is the algorithm of Myers and Miller (1988) CABIOS 4:11-17; The Local Alignment algorithm of Smithet al. (1981) Adv.Appl.Math.2:482; The overall comparison algorithm of Needleman and Wunsch (1970) J.Mol.Biol.48:443-453; The Local Search comparison method of Pearson and Lipman (1988) Proc.Natl.Acad.Sci.85:2444-2448; As Karlin and Altschul (1993) Proc.Natl.Acad.Sci.USA 90:5873-5877 the algorithm of improved Karlin and Altschul (1990) Proc.Natl.Acad.Sci.USA 872264.

Can utilize the PC Tools of these mathematical algorithms, with comparative sequences, thereby determine sequence identity.Described instrument includes but not limited to, the CLUSTAL of PC/ gene program (can obtain from Intelligenetics Mountain View, California); The GAP of ALIGN program (2.0 editions) and GCG Wisconsin genetics software package (the 10th edition), BESTFIT, BLAST, FASTA and TFASTA (can obtain the Inc. from Accelrys, 9685 Scranton Road, San Diego, California, USA).Can use default parameters to utilize these programs to compare.CLUSTAL program is specified in Higgins et al. (1988) Gene 73:237-244 (1988); Higgins et al. (1989) CABIOS 5:151-153; Corpet et al. (1988) Nucleic Acids Res.16:10881-90; Huang et al. (1992) CABIOS 8:155-65; And Pearson et al. (1994) Meth.Mol.Biol.24:307-331.The algorithm of ALIGN program based on above-mentioned Myers and Miller (1988).While using ALIGN program comparision aminoacid sequence, can adopt PAM120 weight residue table, 12 gap length point penalty and 4 interval point penalty.The algorithm of the blast program of Altschul et al. (1990) J.Mol.Biol.215:403 based on above-mentioned Karlin and Altschul (1990).Can carry out BLAST nucleotide search with BLASTN program, score value=100, word length=12, thus the nucleotide sequence of the nucleotide sequence homology of the albumen of acquisition and coding embodiment.Can carry out the search of BLAST albumen with BLASTX program, score value=50, word length=3, thereby obtain and the albumen of embodiment or the aminoacid sequence of homologous peptide.Can be as described in Altschul et al. (1997) Nucleic Acids Res.25:3389, using (BLAST's 2.0) interval BLAST to obtain is relatively the interval comparison of object.Selectively, can use (BLAST's 2.0) PSI-BLAST to carry out the iterative search of source far away relation between detection molecules.Referring to above-mentioned Altschul et al..While using BLAST, interval BLAST, PSI-BLAST, can use the default parameters of program separately (for example, for the BLASTN of nucleotide sequence, for the BLASTX of albumen).Referring to the state-run biotechnology information center website under World Wide Web ncbi.hlm.nih.gov.Can also carry out manual comparison by inspection.

Except as otherwise noted, sequence identity/similarity provided herein represents to use the 10th edition value obtaining of GAP, it uses following parameters: for % consistence and the % similarity of nucleotide sequence, and the GAP weight of its use 50 and 3 length weight, and nwsgapdna.cmp rating matrix; For % consistence and the % similarity of aminoacid sequence, the GAP weight of its use 8 and 2 length weight, and BLOSUM62 rating matrix; Or its any equivalence program.As used herein, term " equivalence program " refer to arbitrary sequence comparison program, it generates comparison for discussed any two sequences, and described comparison has corresponding identical Nucleotide or amino-acid residue coupling and the identical sequence identity per-cent compared generating with GAP the 10th edition.

GAP is used the algorithm of above-mentioned Needleman and Wunsch (1970), to find the comparison to two complete sequence that maximizes coupling number and minimized intervals number.GAP has considered all possible comparison and interval location, and generates the comparison with the minimum interval of maximum match alkali cardinal sum.It allows to provide the interval of coupling base unit to generate point penalty and interval extension point penalty.For each interval of its insertion, GAP must utilize the interval of coupling to generate point penalty number.If select to be greater than 0 interval extension point penalty,, for the interval of each insertion, GAP must utilize gap length to seize an opportunity every extending point penalty in addition.For protein sequence, the default interval of GCG Wisconsin genetics software package (the 10th edition) generates point penalty value and interval extension point penalty value is respectively 8 and 2.For nucleotide sequence, it is 50 that this default interval generates point penalty, and default interval extension point penalty is 3.Interval generates point penalty and interval extension point penalty can be expressed as the integer that is selected from 0 to 200.Thereby for example, interval generates point penalty and interval extension point penalty can be 0,1,2,3,4,5,6,7,8,9,10,15,20,25,30,35,40,45,50,55,60,65 or larger.

GAP represents a best member who compares in family.May there be many members in this family, but other members' quality is all not so good as GAP.GAP shows that 4 of comparison are worth numeral: quality (Quality), ratio (Ratio), consistence and similarity.Quality is maximized tolerance for aligned sequences.Ratio is quality divided by compared with the number of base in short-movie section.Consistence per-cent is the per-cent of the symbol of actual match.Similarity per-cent is the per-cent of similarity sign.Ignore the symbol of crossing over interval.When the right rating matrix value of symbol is more than or equal to 0.50 (similarity threshold value), similarity is marked.The 10th edition score matrix used of GCG Wisconsin genetics software package is BLOSUM62 (referring to Henikoff and Henikoff (1989) Proc.Natl.Acad.Sci.USA 89:10915).

(c) as used herein, when " sequence identity " or " consistence " in two nucleic acid or peptide sequence context refers to compare maximum correspondence in the comparison window in appointment, identical residue in two sequences.For albumen, when using sequence identity per-cent, will be appreciated that inconsistent residue position is often because conserved amino acid replaces different, wherein amino-acid residue is used to replace other amino-acid residues with similar chemical property (as electric charge or hydrophobicity), and does not therefore change the functional property of this molecule.When sequence is different aspect conservative replacement, can raise sequence identity per-cent, to proofread and correct the conservative characteristic of replacement.By this class conservative property replacement, different sequences is considered to have " sequence similarity " or " similarity ".The means of carrying out this adjustment are conventionally known to one of skill in the art.Conventionally, this relates to conservative property is replaced to scoring for part rather than mispairing completely, thereby improves sequence identity per-cent.Therefore, for example, if consistent amino acid whose score is given as to 1, and the score that non-conservation is replaced is given as 0, and the score conservative property being replaced is given as 0 to 1.As performed in program PC/GENE (Intelligenetics, Mountain View, California), calculate conservative property and replace score.

(d) as used herein, " sequence identity per-cent " represents by with respect to comparison window and two right sequences of optimum ratio of comparison and definite value, wherein, for optimum ratio is to two sequences, in comparison window, the part of polynucleotide sequence is compared with reference sequences (it does not comprise interpolation or disappearance), can comprise and add or disappearance (being interval).Per-cent calculates by following: the number of determining the position that nucleic acid base consistent in two sequences or amino-acid residue all exist, thereby draw the number of matched position, number by total number of positions in comparison window except matched position, and result is multiplied by 100, thereby draws sequence identity per-cent.

(e) (i) " the basically identical property " of term polynucleotide sequence represents that polynucleotide comprise while utilizing one of the described comparison program of using canonical parameter, has at least 70%, 80%, 90% or 95% or the sequence of higher sequence identity with reference sequences.One skilled in the art will realize that and can suitably adjust these values, to determine the corresponding consistence of two coded albumen of nucleotide sequence, described definite considered codon degeneracy, amino acid similarity, frame location (reading frame positioning) etc.For the basically identical property of the aminoacid sequence ordinary representation of these objects, there is at least 60%, 70%, 80%, 90% or 95% sequence identity or higher sequence identity.

If two molecule phase mutual crosses under stringent condition, also illustrate that nucleotide sequence is basically identical.Conventionally, under definite ionic strength and pH, select stringent condition, make it than the T of particular sequence _mlow approximately 5 ℃.Yet stringent condition comprises and compares T _mlow approximately 1 ℃ of temperature to 20 ℃ of scopes, this depends on institute as other in this paper required stringency qualitatively.If the coded polypeptide of the nucleic acid of phase mutual cross is not basically identical under stringent condition, described nucleic acid is still basically identical.This can appear at, for example, and when the nucleic acid producing when the maximum codon degeneracy that uses genetic code to allow copies.If the polypeptide of the polypeptide of the first nucleic acid encoding and the second nucleic acid encoding has immune cross-reactivity, illustrate that so two nucleotide sequences are basically identical.

(e) (ii) the contextual term of peptide " basically identical property " is illustrated in the comparison window of appointment, and peptide comprises with reference sequences and has at least 70%, 80%, 85%, 90%, 95% or the conforming sequence of higher sequence.Use the overall comparison algorithm of above-mentioned Needleman and Wunsch (1970), can carry out the optimum ratio pair for these objects.A peptide has immunoreactivity with the antibody of preparing for another peptide, illustrates that two peptide sequences are basically identical.Thereby peptide and another peptide are basically identical, for example, wherein these two peptides are only because of the conservative difference that replaces." substantially similar " peptide has sequence as above, except inconsistent residue position can change different because of conserved amino acid.

Term " constructs " is not intended to embodiment to be limited to the constructs that comprises DNA in use herein.Those of skill in the art will recognize that constructs, the polynucleotide and the oligonucleotide that particularly the combination of ribonucleotide and ribonucleotide and deoxyribonucleotide, consist of also can be for methods disclosed herein.The constructs of embodiment, nucleic acid and nucleotide sequence comprise all complementary types of described construct, molecule and sequence in addition.In addition, the constructs of embodiment, nucleic acid molecule and nucleotide sequence comprise can be for all constructs, molecule and the sequence of the conversion of plant method of embodiment, those that include but not limited to consist of deoxyribonucleotide, ribonucleotide and combination thereof.Described deoxyribonucleotide and ribonucleotide comprise naturally occurring molecule and synthetic analogues.The constructs of embodiment, nucleic acid and nucleotide sequence also comprise the form of ownership of constructs, include but not limited to single stranded form, double chain form, hair clip, loop-stem structure etc.

Another embodiment relates to the organism of conversion, such as the organism that is selected from plant and insect cell, bacterium, yeast, baculovirus, protozoon, nematode and algae.The organism transforming comprises: the DNA molecular of embodiment, the expression cassette that comprises described DNA molecular or the carrier that comprises described expression cassette, it can stably be integrated into the genome of institute's inverting biological body.

The sequence of embodiment is provided in DNA construct, and described construct is for expressing object organism.Described construct comprises 5 ' and 3 ' the modulability sequence that the sequence with embodiment is operatively connected.Term used herein " is operatively connected " the functional connection representing between promotor and another sequence, and the initial and mediation of wherein said promoter sequence is transcribed corresponding to the DNA sequence dna of described another sequence.Conventionally, being operatively connected and representing that the nucleotide sequence be connected is adjacency, and when needs connect two protein-coding regions, is adjacency and in identical frame.Described construct can comprise in addition at least one and by cotransformation, be entered the other gene of organism.Selectively, can in many DNA construct, provide other one or more genes.

Described DNA construct has a plurality of for inserting the restriction site of Cry toxalbumin sequence, and described Cry toxalbumin sequence will be positioned under the transcriptional regulatory in modulability region.Described DNA construct can comprise selectable marker gene in addition.

By 5 ' to 3 ' transcriptional orientation, DNA construct comprises: transcribe with the DNA sequence dna of translation initiation district (being promotor), embodiment and as having the transcribing with translation termination district (being terminator) of function in host's organism.Transcription initiation region (being promotor), with respect to the sequence of host organisms and/or embodiment, can be homology, analogue, external source or allos.In addition, described promotor can be native sequences or be selectively composition sequence.Term used herein " external source " is illustrated in the homology organism that imports promotor and does not find this promotor.When described promotor is " external source " or " allos " with respect to the sequence of embodiment, be intended to show that this promotor is not homology or the naturally occurring promotor of the sequence that is operatively connected in embodiment.As used herein, mosaic gene comprises the encoding sequence being operatively connected with transcription initiation region, and described transcription initiation region is allos for described encoding sequence.When promotor is homology or natural sequence, the expression of the sequence being operatively connected transformation is expressed from wild-type, and this causes the change of phenotype.

Terminator can be homology with transcription initiation region, can be homology with the target DNA sequence being operatively connected, can be homology with plant host, or can derive from another source (that is, with respect to promotor, aim sequence, plant host or its arbitrary combination be external source or allos).

Can obtain terminator easily from agrobacterium tumefaciens (A.tumefaciens) Ti-plasmids, such as octopine synthase and nopaline synthase terminator.Separately see Guerineau et al. (1991) Mol.Gen.Genet.262:141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev.5:141-149; Mogen et al. (1990) Plant Cell2:1261-1272; Munroe et al. (1990) Gene 91:151-158; Ballas et al. (1989) Nucleic Acids Res.17:7891-7903; With Joshi et al. (1987) Nucleic Acid Res.15:9627-9639.

In the time of suitably, can optimize nucleic acid to improve the expression in host organisms.Thereby, if host organisms is plant, can, with the synthetic nucleic acid of favorite plant codon, thereby improve, express.Referring to, for example, Campbell and Gowri (1990) Plant Physiol.92:1-11, the discussion that host's preference codon is used.For example, although all can express the nucleotide sequence of embodiment in monocotyledons and dicotyledons species, but can modification sequence, to solve monocotyledons or special codon preference and the GC content preference of dicotyledons, this is because these preferences have shown difference (Murray et al. (1989) Nucleic Acids Res.17:477-498).Thereby the corn preference codon of specific amino acids can be derived from the known sequence of corn.Article 28, the corn codon of maize plant gene uses and lists in the table 4 of Murray et al. above.This area can obtain the method for synthetic favorite plant gene.Referring to, for example, United States Patent (USP) the 5th, 380, No. 831 and the 5th, 436, No. 391, and Murray et al. (1989) Nucleic Acids Res.17:477-498, it is incorporated to herein by reference.

Known other sequence modification strengthens the genetic expression of cell host.These comprise removing sequence, and the poly-adenosine signal of described sequence encoding vacation, exon-intron splice site signal, swivel base increment repeat and may be unfavorable for the sequence of other well-characterized of genetic expression.The GC content of sequence can be adjusted to the mean level (ML) of given cell host, it calculates with reference to the known at this host cell expression.Term used herein " host cell " refers to comprise carrier and support the cell that this expression vector copies and/or expresses.Host cell can be such as colibacillary prokaryotic cell prokaryocyte, or such as the eukaryotic cell of yeast, insect, batrachians or mammalian cell or monocotyledons or dicotyledons cell.The example of monocotyledons host cell is corn host cell.If possible, modification sequence with avoid expection hair clip mRNA secondary structure.

Expression cassette can comprise 5 ' leader sequence in addition.Described leader sequence can be brought into play the effect that strengthens translation.Translation leader sequence is known in the art and comprises: picornavirus leader sequence, for example EMCV leader sequence (encephalomyocarditis 5 ' non-coding region) (Elroy-Stein et al. (1989) Proc.Natl.Acad.Sci.USA 86:6126-6130); Marmor upsilon leader sequence, TEV leader sequence (tobacco etch virus) (Gallie et al. (1995) Gene 165 (2): 233-238) for example, MDMV leader sequence (the short and small mosaic virus of corn), human immunoglobulin heavy chain is in conjunction with albumen (BiP) (Macejak et al. (1991) Nature 353:90-94); The untranslated leader of alfalfa mosaic virus coat protein mRNA (AMV RNA 4) (Jobling et al. (1987) Nature 325:622-625); Tobacco mosaic virus (TMV) leader sequence (TMV) (Gallie et al. (1989) in Molecular Biology of RNA (molecular biology of RNA), ed.Cech (Liss, New York), pp.237-256); And the sallow mottle virus leader sequence of corn (MCMV) (Lommel et al. (1991) Virology 81:382-385).Separately see Della-Cioppa et al. (1987) Plant Physiol.84:965-968.

When preparing expression cassette, can operate various DNA fragmentations with proper orientation is provided and the DNA sequence dna of suitable frame suitably time.For reaching this purpose, can adopt joint or connexon to connect DNA fragmentation, or can relate to other restriction site that operates to provide convenience, remove DNA redundant, remove restriction site etc.For this purpose, can relate to vitro mutagenesis, primer reparation, restricted, annealing, then replace for example conversion (transition) and transversion (transversion).

When putting into practice embodiment, can use a large amount of promotors.Can select promotor based on results needed.Can make nucleic acid and composing type, organize preference type, induction type or other promotor combination for expressing at host organisms.For the suitable constitutive promoter of plant host cell, comprise, for example, the core promoter of Rsyn7 promotor and WO 99/43838 and United States Patent (USP) the 6th, 072, No. 050 disclosed other constitutive promoter; Core CaMV 35S promoter (Odell et al. (1985) Nature 313:810-812); Rice actin (McElroy et al. (1990) Plant Cell 2:163-171); Ubiquitin (Christensen et al. (1989) Plant Mol.Biol.12:619-632 and Christensen et al. (1992) Plant Mol.Biol.18:675-689); PEMU (Last et al. (1991) Theor.Appl.Genet.81:581-588); MAS (Velten et al. (1984) EMBO J.3:2723-2730); ALS promotor (United States Patent (USP) the 5th, 659, No. 026) etc.Other constitutive promoter comprises, for example, is disclosed in United States Patent (USP) the 5th, 608, No. 149; The 5th, 608, No. 144; The 5th, 604, No. 121; The 5th, 569, No. 597; The 5th, 466, No. 785; The 5th, 399, No. 680; The 5th, 268, No. 463; The 5th, 608, No. 142; With the 6th, those of 177, No. 611.

From inducible promoter expressing gene, can be useful, this depends on results needed.For regulating the nucleotides sequence of embodiment to be listed in the expression in plant, wound induction type (wound-inducible) promotor has special benefit.The damage that described wound inducible promoter can cause insect's food-taking produces reaction, and comprises potato proteinase inhibitor (pin II) gene (Ryan (1990) Ann.Rev.Phytopath.28:425-449; Duan et al. (1996) Nature Biotechnology 14:494-498); Wun1 and wun2, United States Patent (USP) the 5th, 428, No. 148; Win1 and win2 (Stanford et al. (1989) Mol.Gen.Genet.215:200-208); Systemin (McGurl et al. (1992) Science 225:1570-1573); WIP1 (Rohmeier et al. (1993) Plant Mol.Biol.22:783-792; Eckelkamp et al. (1993) FEBS Letters 323:73-76); (Corderok et al. (1994) Plant is (2) J.6: 141-150) for MPI gene; Etc., it is incorporated to herein by reference.

In addition, can in the method for embodiment and constructs, adopt pathogen-inducible promoter.Described pathogen-inducible promoter comprises that it is induced after being subject to pathogenic infection from those of the associated protein (PR albumen) of causing a disease; For example, PR albumen, SAR albumen, beta-1,3-glucanase, chitinase etc.Referring to, for example, Redolfi et al. (1983) Neth.J.Plant Pathol.89:245-254; Uknes et al. (1992) Plant Cell 4:645-656; And VanLoon (1985) Plant Mol.Virol.4:111-116.Separately see WO 99/43819, it is incorporated to herein by reference.

Near Huo Gai position, pathogenic infection position, the promotor of local expression is noticeable.Referring to, Marineau et al. (1987) Plant Mol.Biol.9:335-342 for example; Matton et al. (1989) Molecular Plant-MicrobeInteractions (interaction of the molecular level of plant-microorganism) 2:325-331; Somsisch et al. (1986) Proc.Natl.Acad.Sci.USA 83:2427-2430; Somsisch et al. (1988) Mol.Gen.Genet.2:93-98; And Yang (1996) Proc.Natl.Acad.Sci.USA 93:14972-14977.Separately see Chen et al. (1996) Plant J.10:955-966; Zhang et al. (1994) Proc.Natl.Acad.Sci.USA 91:2507-2511; Warner et al. (1993) Plant J.3:191-201; Siebertz et al. (1989) Plant Cell 1:961-968; United States Patent (USP) the 5th, 750, No. 386 (nematode inducible); And these reference are by being quoted herein.The inducible promoter of corn PRms gene is noticeable especially, its expression be subject to pathogenic agent fusarium moniliforme (Fusarium moniliforme) induction (referring to, for example, Cordero et al. (1992) Physiol.Mol.Plant Path.41:189-200).

Can use chemical to regulate promotor, by application external source Chemical Regulation thing, carry out the expression of regulatory gene in plant.According to object, determine, promotor can be chemical-induced type promotor, and the wherein application inducible gene expression of chemical, or chemical checks promotor, and wherein the application repressor gene of chemical is expressed.Chemical-induced type promotor is known in the art, and comprise, but be not limited to, corn In2-2 promotor, it is activated by benzenesulfonamide herbicide safener, corn GST promotor, and it is activated by the hydrophobic electrophilic compound as Pre-emergency herbicide, and tobacco PR-1a promotor, it is activated by Whitfield's ointment.Chemical that other is paid close attention to regulate promotor comprise the reactive promotor of steroid (referring to, for example, Schena et al. (1991) Proc.Natl.Acad.Sci.USA 88:10421-10425 and McNellis et al. (1998) Plant be (2) J.14: the glucocorticoid inducible type promotor in 247-257) and tsiklomitsin induction type and tsiklomitsin check type promotor (referring to, for example, Gatz et al. (1991) Mol.Gen.Genet.227:229-237 and United States Patent (USP) the 5th, 814, No. 618 and the 5th, 789, No. 156), it is incorporated to herein by reference.

Can utilize and organize preference promotor that the insecticidal proteins expression target of enhancing is fixed in specified plant tissue.Organize preference promotor comprise discussed below those, Yamamoto et al. (1997) Plant is (2) 255-265 J.12; Kawamata et al. (1997) Plant Cell Physiol.38 (7): 792-803; Hansen et al. (1997) Mol.Gen Genet.254 (3): 337-343; Russell et al. (1997) Transgenic Res.6 (2): 157-168; Rinehart et al. (1996) Plant Physiol.112 (3): 1331-1341; Van Camp et al. (1996) Plant Physiol.112 (2): 525-535; Canevascini et al. (1996) Plant Physiol.112 (2): 513-524; Yamamoto et al. (1994) Plant Cell Physiol.35 (5): 773-778; Lam (1994) Results Probl.Cell Differ.20:181-196; Orozco et al. (1993) Plant MolBiol.23 (6): 1129-1138; Matsuoka et al. (1993) Proc Natl.Acad.Sci.USA90 (20): 9586-9590; With Guevara-Garcia et al. (1993) Plant (3): 495-505 J.4.If necessary, can modify these promotors for weak expression.

Leaf preference promotor is known in the art.Referring to, for example, Yamamoto et al. (1997) Plant is (2): 255-265 J.12; Kwon et al. (1994) Plant Physiol.105:357-67; Yamamoto et al. (1994) Plant Cell Physiol.35 (5): 773-778; Gotor et al. (1993) Plant J.3:509-18; Orozco et al. (1993) Plant Mol.Biol.23 (6): 1129-1138; With Matsuoka et al. (1993) Proc.Natl.Acad.Sci.USA90 (20): 9586-9590.

Root preference or root-specific promoter are known, and can be selected from many promotors that can be obtained by document, or again separated from various compatible species.Referring to, for example, Hire et al. (1992) Plant Mol.Biol.20 (2): 207-218 (Soybean Root specificity glutamine synthetase gene); Keller and Baumgartner (1991) Plant Cell 3 (10): 1051-1061 (the root-specific controlling elementss of French bean GRP 1.8 genes); Sanger et al. (1990) Plant Mol.Biol.14 (3): 433-443 (root-specific promoter of agrobacterium tumefaciens mannopine synthase (MAS) gene); With Miao et al. (1991) Plant Cell 3 (1): 11-22 (full length cDNA clone of coding kytoplasm glutamine synthetase (GS), it expresses in Soybean Root and root nodule).Separately see Bogusz et al. (1990) Plant Cell 2 (7): 633-641, wherein described separated two kinds of root-specific promoters from hemochrome gene, described gene is from the non-pulse family mountain jute (Trematomentosa) of the non-pulse family rough leaf mountain jute (Parasponia andersonii) of fixed nitrogen and the non-fixed nitrogen of being correlated with.The promotor of these genes is connected with beta-glucuronidase reporter gene, and be imported into non-pulse family tobacco (Nicotiana tabacum) and pulse family Root or stem of Littleleaf Indianmulberry (Lotus corniculatus) both, and it is active in two examples, all to have retained root-specific promoter.Leach and Aoyagi (1991) described they to the analysis of the promotor of the high expression level rolC of Agrobacterium rhizogenes (Agrobacterium rhizogenes) and rolD root induction gene (referring to Plant Science (Limerick) 79 (1): 69-76).Their conclusion is enhanser with to organize preference terminator dna separated in these promotors.Teeri et al. (1989) is used the gene fusion with lacZ, the agrobatcerium T-DNA gene of octopine synthase of showing to encode is especially active at tip of a root epidermis, and TR2 ' gene is root-specific in complete plant, Qie Yin leaf texture is injured and stimulated, and this is and kills insect or kill the characteristics combination that the coupling of larva gene especially needs (referring to EMBO J.8 (2): 343-350).TR1 ' the gene merging with nptII (neomycin phosphotransferase II) shows similar feature.Root preference promotor in addition comprises VfENOD-GRP3 gene promoter (Kuster et al. (1995) Plant Mol.Biol.29 (4): 759-772); And rolB promotor (Capana et al. (1994) Plant Mol.Biol.25 (4): 681-691.Separately see U.S. Patent number 5,837,876; 5,750,386; 5,633,363; 5,459,252; 5,401,836; 5,110,732; With 5,023,179.

" seed preference " promotor comprise " seed-specific " promotor (activated those promotors of tool in seed development, such as the promotor of seed storage protein) and " seed germination " promotor (activated those promotors of tool in seed germination) both.Referring to Thompson et al. (1989) BioEssays 10:108, it is incorporated to herein by reference.Described seed preference promotor includes, but not limited to Cim1 (information of phytokinin induction); CZ19B1 (corn 19kDa zein); And milps (myo-Inositol-1-phosphate synthase) (referring to United States Patent (USP) the 6th, 225, No. 529, it is incorporated to herein by reference).γ-zein and Glob-1 are endosperm specificity promoters.For dicotyledons, seed specific promoters includes but not limited to, beans β-Kidney bean albumen, napin, β-companion sphaeroprotein (β-conglycinin), soybean agglutinin, Cruciferae albumen (cruciferin) etc.For monocotyledons, seed specific promoters includes but not limited to corn 15kDa zein, 22kDa zein, 27kDa zein, g-zein, waxy, shrunken 1, shrunken 2, sphaeroprotein 1 etc.Separately see WO00/12733, wherein disclose the seed preference promotor from end1 and end2 gene; It is incorporated to herein by reference.The promotor with particular organization's " preference " expression is higher than the expression degree at least one other plant tissue in the degree of the expression of this tissue.Some organize the promotor performance of preference almost in particular organization, to express specially.

When needs low expression level, can use weak promoter.Conventionally, term as used herein " weak promoter " refer to drive the promotor of encoding sequence low expression level.Low expression level means the level of approximately 1/1000 transcript to approximately 1/100,000 transcript to approximately 1/500,000 transcript.Selectively, will be appreciated that term " weak promoter " thus only also comprise in a few cell and in other cell, do not drive and express the promotor that causes total low expression level.When promotor drives while expressing with unacceptable high level, thereby the part that can lack or modify this promoter sequence reduces expression level.

A little less than described, constitutive promoter comprises, for example the core promoter of Rsyn7 promotor (WO99/43838 and United States Patent (USP) the 6th, 072, No. 050), 35S CaMV core promoter etc.Other constitutive promoter comprises, for example, is disclosed in U.S. Patent number 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142; With 6,177, those of 611; It is incorporated to herein by reference.

Conventionally, expression cassette can comprise the marker gene selected of the transformant for selecting.Utilization can select marker gene to select institute's transformant or tissue.Marker gene comprises the gene of the antibiotics resistance of encoding, such as those of coding neomycin phosphotransferase II (NEO) and hygromix phosphotransferase (HPT), and the gene of conferring herbicide compound resistance, described compound is such as careless ammonium phosphine, bromoxynil, imidazolone and 2,4-dichlorphenoxyacetic acid (2,4-D).The other example of the suitable marker gene selected includes but not limited to, the gene of coding to the resistance of following material, and described material is paraxin (Herrera Estrella et al. (1983) EMBO is J.2:987-992); Methotrexate (Herrera Estrella et al. (1983) Nature 303:209-213; With Meijer et al. (1991) Plant Mol.Biol.16:807-820); Streptomycin sulphate (Jones et al. (1987) Mol.Gen.Genet.210:86-91); Spectinomycin (Bretagne-Sagnard et al. (1996) Transgenic Res.5:131-137); Bleomycin (Hille et al. (1990) Plant Mol.Biol.7:171-176); Sulfonamide (Guerineau et al. (1990) Plant Mol.Biol.15:127-136); Bromoxynil (Stalker et al. (1988) Science 242:419-423); Glyphosate (Shaw et al. (1986) Science 233:478-481; With U. S. application sequence number 10/004,357 and 10/427,692); Grass ammonium phosphine (DeBlock et al. (1987) EMBO J.6:2513-2518).Conventionally referring to, Yarranton (1992) Curr.Opin.Biotech.3:506-511; Christopherson et al. (1992) Proc.Natl.Acad.Sci.USA 89:6314-6318; Yao et al. (1992) Cell 71:63-72; Reznikoff (1992) Mol.Microbiol.6:2419-2422; Barkley et al. (1980) in The Operon, pp.177-220; Hu et al. (1987) Cell 48:555-566; Brown et al. (1987) Cell 49:603-612; Figge et al. (1988) Cell 52:713-722; Deuschle et al. (1989) Proc.Natl.Acad.Sci.USA 86:5400-5404; Fuerst et al. (1989) Proc.Natl.Acad.Sci.USA 86:2549-2553; Deuschle et al. (1990) Science 248:480-483; Gossen (1993) Ph.D.Thesis, University of Heidelberg (Ruprecht-Karls-Universitat Heidelberg Ph D dissertation); Reines etal. (1993) Proc.Natl.Acad.Sci.USA 90:1917-1921; Labow et al. (1990) Mol.Cell.Biol.10:3343-3356; Zambretti et al. (1992) Proc.Natl.Acad.Sci.USA 89:3952-3956; Baim et al. (1991) Proc.Natl.Acad.Sci.USA 88:5072-5076; Wyborski et al. (1991) Nucleic Acids Res.19:4647-4653; Hillenand-Wissman (1989) Topics Mol.Struc.Biol.10:143-162; Degenkolb et al. (1991) Antimicrob.Agents Chemother.35:1591-1595; Kleinschnidt et al. (1988) Biochemistry 27:1094-1104; Bonin (1993) Ph.D.Thesis, University of Heidelberg (Ruprecht-Karls-Universitat Heidelberg Ph D dissertation); Gossen et al. (1992) Proc.Natl.Acad.Sci.USA 89:5547-5551; Oliva et al. (1992) Antimicrob.Agents Chemother.36:913-919; Hlavka et al. (1985) Handbook of Experimental Pharmacology (experimental pharmacology handbook), Vol.78 (Springer-Verlag, Berlin); With Gill et al. (1988) Nature 334:721-724.Described open being incorporated to by reference herein.

The listed marker gene of selecting is not intended to limit above.Embodiment can be used arbitrary marker gene of selecting.

The method of embodiment relates to polypeptide or polynucleotide importing plant." importing " be intended to represent to present polynucleotide or polypeptide to plant, so that this sequence enters the cell interior of described plant.The method of embodiment does not rely on by the specific method of polynucleotide or polypeptide importing plant, as long as described polynucleotide or polypeptide enter the inside of at least one cell of described plant.The method of polynucleotide or polypeptide importing plant is known in the art, includes but not limited to stable conversion method, instantaneous conversion method and virus-mediated method.

" stable conversion " is intended to represent that the constructs that imports plant is integrated into Plant Genome, and can entail its filial generation." instantaneous conversion " be intended to represent that polynucleotide are imported into plant but unconformability is entered Plant Genome, or polypeptide is imported into plant.

Conversion operation step and nucleotide sequence is imported to the operation steps of plant can be because of different as transforming the plant of target or the type of vegetable cell (being monocotyledons or dicotyledons).Nucleotide sequence is imported to the proper method that vegetable cell inserts this Plant Genome then and comprise microinjection (Crossway et al. (1986) Biotechniques 4:320-334), electroporation (Riggs et al. (1986) Proc.Natl.Acad.Sci.USA 83:5602-5606), agriculture bacillus mediated conversion (United States Patent (USP) the 5th, 563, No. 055 and the 5th, 981, No. 840), direct gene transfer (Paszkowski et al. (1984) EMBO J.3:2717-2722), with trajectory Particle Acceleration (referring to, for example, U.S. Patent number 4,945,050; 5,879,918; 5,886,244; With 5,932,782; Tomes et al. (1995) in Plant Cell Tissue, and Organ Culture:Fundamental Methods (vegetable cell, tissue and organ culture: basic methods), ed.Gamborg and Phillips (Springer-Verlag, Berlin); With McCabe et al. (1988) Biotechnology 6:923-926); Transform (WO 00/28058) with Lecl.For Transformation of potato, referring to Tu et al. (1998) Plant Molecular Biology 37:829-838 and Chonget al. (2000) Transgenic Research 9:71-78.Other method for transformation can find in Publication about Document, Weissinger et al. (1988) Ann.Rev.Genet.22:421-477; Sanford et al. (1987) Particulate Science and Technology 5:27-37 (onion); Christou et al. (1988) Plant Physiol.87:671-674 (soybean); McCabe et al. (1988) Bio/Technology 6:923-926 (soybean); Finer and McMullen (1991) InVitro Cell Dev.Biol.27P:175-182 (soybean); Singh et al. (1998) Theor.Appl.Genet.96:319-324 (soybean); Datta et al. (1990) Biotechnology 8:736-740 (paddy rice); Klein et al. (1988) Proc.Natl.Acad.Sci.USA 85:4305-4309 (corn); Klein et al. (1988) Biotechnology 6:559-563 (corn); U.S. Patent number 5,240,855; 5,322,783 and 5,324,646; Klein et al. (1988) Plant Physiol.91:440-444 (corn); Fromm et al. (1990) Biotechnology 8:833-839 (corn); Hooykaas-Van Slogteren et al. (1984) Nature (London) 311:763-764; United States Patent (USP) the 5th, 736, No. 369 (cereal); Bytebier et al. (1987) Proc.Natl.Acad.Sci.USA 84:5345-5349 (Liliaceae); De Wet et al. (1985) in TheExperimental Manipulation of Ovule Tissues (experimental implementation of ovule tissue), ed.Chapman et al. (Longman, New York), pp.197-209 (pollen); Kaeppler et al. (1990) Plant Cell Reports 9:415-418 and Kaeppler et al. (1992) Theor.Appl.Genet.84:560-566 (conversion of whisker mediation); D ' Halluin et al. (1992) Plant Cell 4:1495-1505 (electroporation); Li et al. (1993) Plant Cell Reports 12:250-255 and Christou and Ford (1995) Annals of Botany 75:407-413 (paddy rice); Osjoda et al. (1996) Nature Biotechnology 14:745-750 (corn passes through agrobacterium tumefaciens); All being all incorporated to by reference herein above.

In embodiment, can use various instantaneous conversion methods that the sequence of embodiment is provided to plant.Described instantaneous conversion method includes but not limited to, Cry toxin protein or its variant is directly imported to plant with its fragment, or Cry toxin transcript is imported to described plant.Described method comprises, for example, and microinjection or partickle bombardment.Referring to, for example, Crossway et al. (1986) Mol Gen.Genet.202:179-185; Nomura et al. (1986) Plant Sci.44:53-58; Hepler et al. (1994) Proc.Natl.Acad.Sci.91:2176-2180 and Hush et al. (1994) The Journal of Cell Science 107:775-784, above all being all incorporated to by reference herein.Selectively, use technology known in the art the polynucleotide instantaneous conversion of Cry toxin can be entered to plant.Described technology comprises virus carrier system and the precipitation of polynucleotide is consequently stoped to DNA release subsequently.Thereby, can occur, from the transcribing of the DNA of particle combination, but it is released, to be integrated into genomic frequency and greatly to reduce.Described method comprises uses the poly-coated particle (PEI of ethyliminum; Sigma #P3143).

The method of inserting polynucleotide at Plant Genome specific position target known in the art.In one embodiment, use site-specific recombination system to realize and in required genome position, insert polynucleotide.Referring to, for example, WO99/25821, WO99/25854, WO99/25840, WO99/25855 and WO99/25853, above all being all incorporated to by reference herein.In brief, the polynucleotide of embodiment can be included in transfer box, and described transfer box flank connects two incomparable inconsistent recombination sites.This transfer box is imported to plant, in target site stable integration, enter its genome, described target site flank connects two incomparable inconsistent recombination sites corresponding to transfer box site.Suitable recombinase is provided, and transfer box is integrated in target site.Thereby polynucleotide of interest specific chromosome position in Plant Genome is integrated.

According to usual manner, can make the Growth of Cells being converted become plant.Referring to, for example, McCormick et al. (1986) Plant Cell Reports 5:81-84.Can make these plant-growths subsequently, and with identical conversion strain or different lines pollination, identify and there is the composing type of desired phenotype feature or the gained heterozygote of inducible expression.Can grow two generations or more generations, thereby guarantee that the expression of desired phenotype feature keeps stable and quilt heredity, gathers in the crops seed, subsequently to guarantee to realize the expression of desired phenotype feature.

Can, by plant is contacted with virus or viral nucleic acid, to plant, provide the nucleotide sequence of embodiment.Conventionally, described method relates in viral DNA or RNA molecule and is incorporated to object constructs.It should be understood that the part using the recombinant protein of embodiment as viral polymeric protein is synthesized at first, subsequently can by body or external proteolysis process described polymeric protein, thereby produce required insecticidal proteins.This viral polymeric protein that also will be appreciated that at least a portion of the aminoacid sequence that comprises embodiment insecticidal proteins can have required insecticidal activity.Embodiment comprises described viral polymeric protein and encodes their nucleotide sequence.The method that constructs is provided and produces coded albumen in plant to plant is known in the art, and it relates to viral DNA or RNA molecule.Referring to, for example, U.S. Patent number 5,889,191; 5,889,190; 5,866,785; 5,589,367; With 5,316,931; It is incorporated to herein by reference.

Embodiment also relates to the plant propagation material of institute's conversion of plant of embodiment, includes but not limited to seed, stem tuber, bulb, bulb, leaf, and the cutting of root and bud.

Embodiment can, for transforming any plant species, include but not limited to monocotyledons and dicotyledons.The example of the plant of paying close attention to includes but not limited to corn (Zea mays), the kind of Btassica (Brassica) is (as rape (B.napus), turnip (B.rapa), leaf mustard (B.juncea)), particularly can be used as the kind of those Btassicas in seed oil source, clover (Medicago sativa), paddy rice (Oryza sativa), rye (Secale cereale), Chinese sorghum (Sorghum bicolor, Sorghum vulgare), grain is (as Herba penniseti (Pennisetum glaucum), broomcorn millet (Panicum miliaceum), millet (Setaria italica), Finger-millet (Eleusine coracana)), Sunflower Receptacle (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanut (Arachis hypogaea), cotton (sea island cotton (Gossypium barbadense), upland cotton (Gossypium hirsutum)), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Coffea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana), Fructus Fici (Ficus casica), piscidia (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), pawpaw (Carica papaya), cashew nut (Anacardium occidentale), nut (Macadamia integrifolia), apricot (Prunus amygdalus), beet (Beta vulgaris), sugarcane (Saccharum spp.), oat, barley, vegetables, ornamental plant and softwood tree.

Vegetables comprise tomato (Lycopersicon esculentum), lettuce (Lactuca sativa), Kidney bean) Phaseolus vulgaris), the member of lima bean (Phaseolus limensis), pea (Lathyrus spp.) and Cucumis (Cucumis) is as cucumber (C.sativus), cantaloupe (C.cantalupensis) and muskmelon (C.melo).Ornamental plant comprises rhododendron (Rhododendron spp.), Flower of Largeleaf Hydrangea (Macrophylla hydrangea), the rose of Sharon (Hibiscus rosasanensis), rose (Rosa spp.), turmeric (Tulipa spp.), flower of Chinese Narcissus (Narcissus spp.), petunia (Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia (Euphorbia pulcherrima) and chrysanthemum.The softwood tree that can be used for putting into practice embodiment for example comprises: pine, such as torch pine (Pinus taeda), slash pine (Pinus elliotii), ponderosa pine (Pinus ponderosa), black pine (Pinus contorta) and pine (Pinus radiata); Pseudotsuga menziesii (Mirbel) Franco (Pseudotsuga menziesii); Canadian hemlock (Tsuga canadensis); White spruce (Picea glauca); Chinese larch (Sequoia sempervirens); True fir, such as silver-colored fir (Abies amabilis) and balsam fir (Abies balsamea); And cdear, such as the red cdear (Thuja plicata) in west and Alaska golden cypress (Chamaecyparis nootkatensis).The plant of embodiment comprises farm crop (such as corn, clover, Sunflower Receptacle, Btassica, soybean, cotton, safflower, peanut, Chinese sorghum, wheat, grain, tobacco etc.), such as corn and soybean plants.

Turfgrass includes but not limited to annual annual bluegrass (Poa annua); Annual ryegrass (Lolium multiflorum); Canada blue grass (Poa compressa); Qiu Shi fescue grass (Festuca rubra); Thin and delicate creeping bentgrass (Agrostis tenuis); Creeping bentgrass (Agrostispalustris); Crested wheatgrass (Agropyron desertorum); Crested wheat grass (Agropyron cristatum); Hard fescue (Festuca longifolia); English grass (Poa pratensis); Orchard grass (Dactylis glomerata); English ryegrass (Lolium perenne); Red fescue (Festuca rubra); White bent (Agrostis alba); Rough bluegrass (Poa trivialis); Fescue grass (Festuca ovina); Awnless brome (Bromus inermis); Festuca Arundinacea (Festuca arundinacea); Thimothy grass (Phleum pratense); Velvet bent grass (Agrostis canina); Alkali thatch (Puccinellia distans); Blue stem ice grass (Agropyron smithii); Bermuda grass (Cynodon spp.); Saint augustine grass (Stenotaphrum secundatum); Jielu grass (Zoysia spp.); Bahia grass (Paspalum notatum); Carpetweed (Axonopus affinis); Eremochloa ophiuroides (Eremochloa ophiuroides); Herba penniseti (Pennisetum clandesinum); Seashore paspalum (Paspalum vaginatum); Gramagrass (Bouteloua gracilis); Buffalo grass (Buchloe dactyloids); Side fringe gramagrass (Bouteloua curtipendula).

The plant of paying close attention to comprises cereal grass, oleaginous seed plant and the leguminous plants of the seed that provides paid close attention to.The seed of paying close attention to comprises seed corn, as corn, wheat, barley, paddy rice, Chinese sorghum, rye, broomcorn millet etc.Oleaginous seed plant comprises cotton, soybean, safflower, Sunflower Receptacle, Btassica, corn, clover, palm, coconut, flax, castor-oil plant, olive etc.Leguminous plants comprises Kidney bean and pea.Kidney bean comprises guar-bean, locust bean, Semen Trigonellae, soybean, French bean, cowpea, mung bean, lima bean, broad bean, root of Szemao crotalaria, garbanzo etc.

In some embodiments, the nucleotide sequence of embodiment can be stacking with the polynucleotide of interest sequence of arbitrary combination, thereby produce the plant with desired phenotype.For example, the polynucleotide of embodiment can have desinsection with coding and/or kill other any polynucleotide of polypeptide of insect active stacking, and described polypeptide (is described in U.S. Patent number 5,366,892 such as other Bt toxin protein; 5,747,450; 5,736,514; 5,723,756; 5,593,881; With Geiser et al. (1986) Gene 48:109), pentin (being described in United States Patent (USP) the 5th, 981, No. 722) etc.The combination generating can also comprise arbitrary polynucleotide of interest of multiple copied.Also can the combination of the arbitrary gene of the polynucleotide of embodiment and other or gene is mutually stacking, thus the plant with various required proterties combinations produced, and it includes but not limited to that animal takes food required proterties, such as high oil base for example, because of (, United States Patent (USP) the 6th, 232, No. 529); Balanced type amino acid (for example, hordothionins (U.S. Patent number 5,990,389; 5,885,801; 5,885,802; With 5,703,049); Barley high-lysine (Williamson et al. (1987) Eur.J.Biochem.165:99-106; With WO 98/20122) and homomethionine albumen (Pedersen et al. (1986) J.Biol.Chem.261:6279; Kirihara et al. (1988) Gene 71:359; With Musumura et al. (1989) Plant Mol.Biol.12:123)); The digestibility strengthening (for example, the storage protein of modification (the U. S. application sequence number 10/053,410 that submit to November 7 calendar year 2001); And Trx (the U. S. application sequence number 10/005,429 that submit to December 3 calendar year 2001), its disclosure is incorporated to herein by reference.

Can also for example, by the polynucleotide of embodiment and disease or required mutually stacking (, the FT detoxification gene (United States Patent (USP) the 5th, 792, No. 931) of proterties of Herbicid resistant; Nontoxicity and disease resistance gene (Jones et al. (1994) Science 266:789; Martin et al. (1993) Science 262:1432; With Mindrinos et al. (1994) Cell 78:1089); Acetolactate synthase (ALS) mutant that causes Herbicid resistant, such as S4 and/or Hra sudden change; Glutamine synthase inhibitor, for example, such as careless ammonium phosphine or basta (, bar gene); And glyphosate resistance (EPSPS gene and GAT gene, as U. S. application sequence number 10/004,357; With 10/427,692 disclosed); And mutually stacking with processing or the required proterties of processed products, for example, such as high oil (, United States Patent (USP) the 6th, 232, No. 529); Oil (for example, fatty acid desaturase gene (United States Patent (USP) the 5th, 952, No. 544 of modifying; WO 94/11516)); The starch of modifying (for example, ADPG pyrophosphorylase (AGPase), starch synthase (SS), starch branching enzyme (SBE) and starch remove branching enzyme (SDBE)); And polymer or biological plastics (for example, United States Patent (USP) the 5.602nd, No. 321; β-ketothiolase; poly(hydrobutyl ester) synthase; and acetoacetyl-CoA reductase enzyme (Schubert et al. (1988) J.Bacteriol.170:5837-5847) promotes the expression of polyhydroxyalkanoatefrom (PHA)), its disclosure is incorporated to herein by reference.Also can be by the polynucleotide of embodiment and the polynucleotide combination that Agronomic character or transformation technology proterties are provided, described Agronomic character such as male sterile (for example, referring to United States Patent (USP) the 5.583rd, No. 210), stem strength, flowering time, (for example, WO 99/61619 for described transformation technology proterties such as Cycle Regulation or gene targeting; WO 00/17364; WO 99/25821), its disclosure is incorporated to herein by reference.

Can be by including but not limited to that any method of cross-breeding plant or genetic transformation produces these stacking compositions, described cross-breeding plant by any ordinary method or method.If carry out stacking proterties by genetic transformation plant, so can be at any time with combined in any order polynucleotide of interest sequence.For example, the transgenic plant that comprise one or more required proterties can be used as to target, thereby import other proterties by conversion subsequently.Can use and transform the polynucleotide of interest that the arbitrary combination of box provides, with cotransformation operation steps, import described proterties simultaneously.For example, if import two sequences, so described two sequences can be included in conversion box (trans) separately or be included in identical conversion box (cis).Can be by identical promotor or by the expression of different promoters driven sequences.In some cases, need to import the conversion box that suppresses polynucleotide of interest expression.The arbitrary combination that can suppress by itself and other box or mistake expression cassette is combined, thereby in plant, generates required proterties combination.Also recognize and can use site-specific recombination system, polynucleotide sequence is stacking in required genome position.Referring to, for example, WO99/25821, WO99/25854, WO99/25840, WO99/25855 and WO99/25853, all these is incorporated to herein by reference.

Can be by the composition of embodiment the plant product for the protection of plant, seed and variety of way.For example, composition can be used for following method, the method relates to the insect-killing composition of significant quantity is placed in to insect environment by operation, and described operation is selected from sprinkling (spraying), dusting (dusting), broadcast sowing (broadcasting) or seed is coated.

Using plant propagation material (fruit, stem tuber, bulb, bulb, cereal, seed), when especially seed is as merchandise sales; conventionally coated by its processing with protective material; described protective material comprises weedicide, insecticide, mycocide, bactericide, nematocides, invertebrate poison; or several mixture in these prepared products; if needed; and process with other carrier, tensio-active agent or the short application adjuvant that is generally used for formulation art, thereby provide the protection of the infringement that bacterium, fungi or animal pest are caused.In order to process seed, can be by with liquid preparation dipping piece root or cereal, or the moist or dryness preparation combining by use is coated with, and protective material is coated with for seed.In addition, under special circumstances, other method that is applied to plant is feasible, for example, for the processing of bud or fruit.

Can be coated with the plant seed of processing embodiment with seed protectant; the nucleotide sequence of the insecticidal proteins that described plant seed comprises the embodiment of encoding; and described seed protectant comprises seed treatment compound; such as; for example, Vancide 89, carboxin, thiram, methalaxyl, Actellic and be generally used for other compound of seed treatment.In one embodiment, the seed protectant of the insect-killing composition that comprises embodiment is coated to be used separately, or a kind of being used in combination in coated with the seed protectant that is generally used for seed treatment.

Recognize and can transform with the gene of encoding insecticidal proteins insect pathogenic organisms body.Described organism comprises baculovirus, fungi, protozoon, bacterium and nematode.

Can the gene of the insecticidal proteins of coding embodiment be imported to microorganism host by suitable carrier, described host is applied to environment or plant or animal.Term " importing " in the context that nucleic acid is inserted to cell refers to " transfection " or " conversion " or " transduction ", and comprise relating to nucleic acid is incorporated to eucaryon or prokaryotic cell prokaryocyte, wherein nucleic acid (for example can be integrated into cellular genome, karyomit(e), plasmid, plastid or Mitochondrial DNA), be transformed into self-replicating, or transient expression (for example, the mRNA of transfection).

Can select the known microorganism host that occupies one or more object crops " phytosphere " (blade face, leaf enclose, rhizosphere and/or root face).Select these microorganisms, can in specific environment, successfully compete with wild-type microorganisms, make to express insecticidal proteins stable gene maintain and express, and satisfactorily, improve the protection to environment degradable and inactivation sterilant.

Described microorganism comprises bacterium, algae and fungi.The all bacteriums in this way of microorganism of noticeable especially concern, for example, Pseudomonas (Pseudomonas), erwinia (Erwinia), serratia (Serratia), klebsiella spp (Klebsiella), xanthomonas (Xanthomonas), streptomyces (Streptomyces), rhizobium (Rhizobium), Rhodopseudomonas (Rhodopseudomonas), Methylius, Agrobacterium (Agrobacterium), genus acetobacter (Acetobacter), lactobacillus genus (Lactobacillus), Arthrobacter (Arthrobacter), Azotobacter (Azotobacter), leuconos toc (Leuconostoc) and Alkaligenes (Alcaligenes), it is fungi, yeast in particular, yeast belong (Saccharomyces) for example, genera cryptococcus (Cryptococcus), Crewe is tieed up this yeast belong (Kluyveromyces), Sporobolomyces (Sporobolomyces), Rhodotorula (Rhodotorula) and black yeast belong to (Aureobasidium).Special concern be phytosphere bacterial species, such as pseudomonas syringae (Pseudomonas syringae), Pseudomonas fluorescens (Pseudomonas fluorescens), serratia marcescens (Serratia marcescens), wood vinegar bacterium (Acetobacter xylinum), Agrobacterium (Agrobacteria), Rhodopseudomonas spheroides (Rhodopseudomonas spheroids), xanthomonas campestris (Xanthomonas campestris), rhizobium melioti (Rhizobium melioti), alcaligenes eutrophus (Alcaligenes entrophus), wooden rod shape bacillus (Clavibacter xyli) and Wei Nielande vinelandii (Azotobacter vinlandir) and phytosphere yeast species, such as rhodothece rubra (Rhodotorula rubra), rhodotorula glutinis (R.glutinis), Rhodotorula marina (R.marina), orange red yeast (R.aurantiaca), shallow white cryptococcus (Cryptococcus albidus), wandering Cryptococcus (C.diffluens), Lauren cryptococcus (C.laurentii), rose yeast (Saccharomyces rosei), S.pretoriensis, yeast saccharomyces cerevisiae (S.cerevisiae), rose-colored shadow yeast (Sporobolomyces roseus), fragrance shadow yeast (S.odorus), Kluyveromyces veronae and Aureobasidium pullulans (Aureobasidium pollulans).Special concern be pigment microorganism.

Can use existing many modes, under the condition that allows stably to maintain with expressing gene, the gene that will express insecticidal proteins imports microorganism host.For example, energy construction expression box, described expression cassette comprise object constructs and with host organisms in sequence homology nucleotide sequence and/or in this host, there is the dubbing system of function, described constructs with for expressing the modulability signal of transcribing and translate of this constructs, be operatively connected, by described nucleotide sequence, integrate, by described dubbing system, occur to integrate or stable maintaining.

Transcribe and translate modulability signal and include but not limited to, promotor, transcription initiation site, operation gene, activator (activator), enhanser, other modulability element, ribosome bind site, initiator codon, termination signal etc.Referring to, for example, United States Patent (USP) the 5th, 039, No. 523 and the 4th, 853, No. 331; EPO 0480762A2; Sambrook et al. (1992) Molecular Cloning:A Laboratory Manual (molecular cloning: laboratory manual), ed.Maniatis et al. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York), call " Sambrook II " in the following text; Davis et al., eds. (1980) Advanced Bacterial Genetics (senior bacterial genetics) (Cold Spring Harbor Laboratory Press), Cold Spring Harbor, New York; And described reference is by being quoted herein.

Suitable host cell can comprise prokaryotic cell prokaryocyte or eukaryotic cell, it is limited to those cells that do not produce such as the virose material of mammiferous higher organism body conventionally, wherein process the cell containing insecticidal proteins, thereby when being subject to processing cell and being applied to there is the environment of one or more target insects, extend the activity of insecticidal proteins in described cell.Yet, can use the organism producing the virose material of higher organism body tool, wherein said toxin is unstable or application level is enough low, so that avoids mammalian hosts to exist any possibility of toxicity.As host, special concern be prokaryotic cell prokaryocyte and the eukaryotic cell such as low, such as fungi.Exemplary prokaryotic cell prokaryocyte (gram-positive microorganism and Gram-negative bacteria) comprises enterobacteriaceae (Enterobacteriaceae), such as Escherichia (Escherichia), erwinia, Shigella (Shigella), salmonella (Salmonella) and proteus (Proteus); Bacillaceae (Bacillaceae); Rhizobiaceae (Rhizobiaceae), such as rhizobium (Rhizobium); Spirillaceae (Spirillaceae), such as luminescent bacteria, Zymomonas (Zymomonas), serratia, Aeromonas (Aeromonas), Vibrio (Vibrio), Desulfovibrio (Desulfovibrio), spiral Pseudomonas (Spirillum); Lactobacillaceae (Lactobacillaceae); Pseudomonadaceae (Pseudomonadaceae) is such as Rhodopseudomonas and acetobacter; Azotobacteraceae (Azotobacteraceae) and Nitrobacteraceae (Nitrobacteraceae).Eukaryotic cell has fungi, and such as phycomycetes (Phycomycetes) and Ascomycetes (Ascomycetes), it comprises yeast, such as yeast belong and Schizosaccharomyces (Schizosaccharomyces); And basidiomycetes (Basidiomycetes) yeast, such as Rhodotorula, aureobasidium genus (Aureobasidium), Sporobolomyces etc.

For producing insecticidal proteins, selecting the feature of special concern aspect host cell to comprise that convenient, the availability of expression system that insecticidal protein gene is imported to host is, the efficiency of expression, the stability of albumen in host and the existence of auxiliary hereditary potency.The feature of paying close attention to as sterilant microcapsule comprises the protectiveness characteristic for sterilant, such as thick cell walls, pigmentation and born of the same parents' internal packing or inclusion body, forms; Leaf affinity; The shortage of mammalian toxicity; The attraction that insect is eaten; Kill and fix and do not damage the convenient of toxalbumin; Etc..Other consideration comprises the convenient of preparation and processing, economy, storage stability etc.

The host organisms of special concern comprises yeast, such as rhodotorula (Rhodotorula spp.), short stalk mould (Aureobasidium spp.), yeast (Saccharomyces spp.) (such as, yeast saccharomyces cerevisiae), shadow yeast (Sporobolomyces spp.); Blade face organism, such as pseudomonas (Pseudomonas spp.) (such as Pseudomonas aeruginosa (P.aeruginosa), Pseudomonas fluorescens), Erwinia (Erwinia spp.) and xanthin bacterium (Flavobacterium spp.); And other this type of organism, comprise Bt, intestinal bacteria, Bacillus subtillis (Bacillus subtilis) etc.

The gene of the insecticidal proteins of coding embodiment can import the microorganism (epiphyte) that relies on plant multiplication, thereby insecticidal proteins is delivered to potential target insect.Epiphyte for example, can be gram-positive microorganism or gram-positive microorganism.

Root is grown the bacterium of (root-colonizing) surely, for example, and can be by the separated plant from paying close attention to of means known in the art.Particularly, surely grow wax-like bacillus (Bacillus cereus) bacterial strain of root can be separated from roots of plants (for example, referring to,, Handelsman et al. (1991) Appl.Environ.Microbiol.56:713-718).Can, by standard method known in the art, the gene of the insecticidal proteins of coding embodiment be imported to the wax-like bacillus that root is grown surely.

By electric conversion energy, the gene of encoding insecticidal proteins is imported, for example, the genus bacillus that root is grown surely.Particularly, the gene of encoding insecticidal proteins can be cloned into shuttle vectors, for example, and pHT3101 (Lerecius et al. (1989) FEMS Microbiol.Letts.60:211-218).The shuttle vectors pHT3101 of the encoding sequence that comprises specific insecticidal protein gene, for example, can be converted into by electroporation the genus bacillus (Lerecius et al. (1989) FEMS Microbiol.Letts.60:211-218) that root is grown surely.

Can design expression system, consequently, for example, insecticidal proteins be secreted to the kytoplasm such as colibacillary Gram-negative bacteria.The advantage of insecticidal proteins secretion is: (1) avoids the potential cytotoxic effect of expressed insecticidal proteins; And (2) improve the purification efficiency of insecticidal proteins, include but not limited to, improve protein renaturation and the purification efficiency of every volume cell meat soup, shorten the time of per unit protein renaturation and purifying and/or reduce its cost.

The insecticidal proteins that can prepare intestinal bacteria secretion, for example, by suitable intestinal bacteria signal peptide is merged to the aminoterminal end to insecticidal proteins.Can in the albumen of the known secretion of intestinal bacteria, find the signal peptide of intestinal bacteria identification, for example OmpA albumen (Ghrayeb et al. (1984) EMBO J, 3:2437-2442).OmpA is the major protein of intestinal bacteria adventitia, its signal peptide thereby be considered in transposition process effectively.In addition, before processing, OmpA signal peptide does not need to be modified, for example other signal peptide possibility (Duffaud et al. (1987) Meth.Enzymol.153:492) like this of lipoprotein signal peptide.

The insecticidal proteins of embodiment can ferment in host bacterium, machining obtain bacterium and by its with by Bt bacterial strain as the same way of killing insect sprays as microorganism sprays.The in the situation that of one or more insecticidal proteins of secreted from bacillus, use means known in the art to remove or sudden change secretion signal.Described sudden change and/or disappearance stop secretes one or more insecticidal proteins to growth medium during the fermentation.In cell, retain insecticidal proteins, process subsequently described cell to produce the insecticidal proteins of encapsulation.The microorganism that can use any appropriate for this object.Pseudomonas has been used to Bt toxin to be expressed as the albumen of encapsulation, machining obtains cell, and spray it as insecticide (Gaertner et al. (1993), in:Advanced Engineered Pesticides (senior through engineering approaches sterilant), ed.Kim).

Selectively, by heterologous gene transfered cell host is produced to insecticidal proteins.The expression of heterologous gene causes directly or indirectly the interior generation of the born of the same parents of sterilant and maintains.While subsequently cell being applied to have the environment of one or more target insects, process these cells extending cell and produce under the condition of toxin activity.Products therefrom retains the toxicity of toxalbumin.Can prepare according to routine techniques the insecticidal proteins of these natural encapsulation subsequently, to be applied to have the environment of target insect, the leaf of soil, water and plant for example.Referring to, for example EPA 0192319, and described reference is by being quoted herein.

In embodiment, acceptable carrier can by the microorganism transforming, (it comprises whole organism, cell, one or more spores, one or more insecticidal proteins, one or more insect disinfestation components, one or more affect the component of insect, one or more mutant, viable cell or dead cell and cellular component, the mixture that comprises viable cell and dead cell and cellular component, and comprise damaged cell and cellular component) or separated insecticidal proteins be mixed with one or more insect-killing compositions, described insect-killing composition, for example suspension, solution, emulsion, pulvis, dispersible particle or bead, wettable powder and emulsifiable concentrate, smoke substance or sprays, impregnated granules, adjuvant, can be coated with paste, colloid, and in addition with for example polymer material encapsulation.Can prepare by conventional means the composition of described preparation, described means such as dry, freeze-drying, homogenize, extract, filtration, centrifugal, sedimentation or the concentrated cell culture that comprises described polypeptide.

Can pass through other prepared product that adds tensio-active agent, inert support, sanitas, wetting agent, feeding stimulant, attractive substance, encapsulants, tamanori, emulsifying agent, dyestuff, UV protective material, damping fluid, flow agent or fertilizer, micro-nutrition donor or affect plant-growth, obtain above disclosed described composition.Can, by one or more agrochemicals and carrier, tensio-active agent or the adjuvant or other combination of components that are generally used for formulation art, take and promote to process and application as the product of particular target insect.Suitable carrier and adjuvant can be solid or liquid, and corresponding to the material that is generally used for preparation technique, for example, mineral substance, solvent, dispersion agent, wetting agent, tackifier, tamanori or fertilizer natural or regeneration.The activeconstituents of embodiment is applied with composition forms conventionally, and can be applied to pending crop region, plant or seed.For example, can be when preparing silo or silo etc., or when the storages such as silo or silo, the composition of embodiment is applied to cereal.Can with other compound simultaneously or the composition of continuous application embodiment.The method of the activeconstituents of application implementation mode or the agricultural chemical composition of embodiment includes but not limited to foliar application, seed is coated and soil application, at least one insecticidal proteins that the bacterial isolates that described activeconstituents or agricultural chemical composition comprise embodiment produces.The intensity that the quantity of application and speed depend on corresponding insect infestation.

Suitable tensio-active agent includes but not limited to anionic compound, such as, metal carboxylate for example; Longer chain fatty acid carboxylate salt; N-acyl sarcosinate; The salt of the monobasic of phosphoric acid and fatty alcohol ethoxylate or dibasic ester or described ester; Aliphatic alcohol sulfate, such as sodium lauryl sulphate, sodium stearyl sulfate or Sodium palmityl sulfate; Fatty alcohol ethoxylate vitriol; Oxyethyl group alkyl phenol vitriol; Sulfonated lignin; Sulfonated petro-leum; Alkylaryl sulphonate, such as alkyl-benzene sulfonate or low alkyl group naphthalenesulfonate, for example, butyl-naphthalenesulfonate; The salt of sulfonated naphthalene-formaldehyde condensation products; The salt of sulfonated phenol-formaldehyde condensation products; More complicated sulfonate, such as, amidosulfonic acid salt, for example the sulfonation condensation product of oleic acid and N-N-methyltaurine; Or dialkyl sulfosuccinates, for example sodium sulfonate of dioctyl succinate.Non-ion reagent comprises that phenol that fatty acid ester, fatty alcohol, fatty acid amide or fat-alkyl or alkenyl replace and the condensation product of oxyethane are, the fatty ester of polyol ethers, for example, sorbitan aliphatic ester, the condensation product of described ester and oxyethane, for example, polyoxyethylene sorbitan aliphatic ester, oxyethane and propylene oxide, alkyne diol are such as 2,4,7,9-tetraethyl--5-decine-4,7-glycol, or the segmented copolymer of oxyethyl group alkyne diol.The example of cats product comprises, for example, aliphatics monoamine, diamine or polyamine such as acetate, naphthenate or oleate; Or oxygen containing amine, such as the amine oxide of polyoxyethylene alkyl amine; The amine of the amide linkage of preparing by condensation carboxylic acid and binary or polyamine; Or quaternary ammonium salt.

The example of inert material includes but not limited to inorganic mineral such as kaolin, phyllosilicate, carbonate, vitriol, phosphoric acid salt or vegetable material such as cork, mealy corn cob, Pericarppium arachidis hypogaeae, rice husk and walnut shell.

The composition of embodiment can be for direct application or as the appropriate form of initial composition enriched material, described enriched material needs to dilute with appropriate water or other thinner before application.Insecticide concentration changes according to the character of particular formulations, particularly, according to it, is enriched material or will directly uses and change.Described composition comprises 1% to 98% solid or liquid inert support, and 0% to 50% or 0.1% to 50% tensio-active agent.With the ratio applying said compositions on Commercial goods labels, every acre of about 0.01lb-5.0lb during dried forms for example, every acre of about 0.01pts.-10pts. during liquid form.

In another embodiment, before preparation, can process the composition of embodiment and the microorganism of conversion and insecticidal proteins, thereby when being applied to the environment of target insect, extend insecticidal activity, as long as pre-treatment does not have harm to insecticidal activity.Can carry out described processing by chemistry and/or physical means, as long as described processing does not deleteriously affect the character of one or more compositions.The example of chemical reagent includes but not limited to halogenating agent; Aldehyde such as formaldehyde and glutaraldehyde; Anti-infection agent, such as Alkyldimethy Ammoni-um Choride; Alcohol, such as Virahol and ethanol; And histological fixative, such as cloth peace fixing agent (Bouin ' s fixative) and extra large Li Shi fixing agent (Helly ' s fixative) (for example, referring to, Humason (1967) Animal Tissue Techniques (animal tissues's technology) (W.H.Freeman and Co.).

In other embodiments, can advantageously use for example tryptic protease treatment Cry toxin polypeptide, thereby activated described albumen before the insecticidal proteins composition of embodiment is applied to target insect environment.Known in this field by the method for serine stretch protein enzyme activation toxogen.Referring to, for example, Cooksey (1968) Biochem.J.6:445-454 and Carroll and Ellar (1989) Biochem.J.261:99-105, be incorporated to its instruction herein by reference.For example, suitable activation act step includes but not limited to polypeptide to be activated, the new Cry polypeptide of purifying (for example, thering is the aminoacid sequence as described in SEQ ID NO:2) for example, with trypsinase at 20nM NaHCO ₃, in pH 8, combined with albumen/trypsinase weight ratio 1/100, and described sample is digested 3 hours at 36C.

Can start to occur or before insect occurs insect; composition (the institute's microbial and the insecticidal proteins that comprise embodiment) is applied to insect pest environment as sfgd.; described application is for example passed through; sprinkling, atomization, efflorescence, distribution, coated or topple over, introduce soil or cause soil surface, introduce irrigation water, it is by seed treatment or common application or efflorescence.For example, the microorganism of the insecticidal proteins of embodiment and/or conversion can be mixed with cereal, thus the cereal of protection shelf time.Conventionally plant importantly in plant-growth, obtains in early days the good control to insect, because now can be subject to the most serious infringement.As thought, be necessary, the composition of embodiment can comprise another insecticide easily.In one embodiment, when plantation, composition is directly applied to soil, described application is the particle form with the combination of the dead cell of carrier and Bacillus strain or embodiment institute microbial.Another embodiment is the particle form of composition, and described composition comprises agrochemicals, such as, for example, the dead cell of institute's microbial of weedicide, insecticide, fertilizer, inert support and Bacillus strain or embodiment.

Those skilled in the art will recognize that, not all compound all has effect same to whole insects.The compound of embodiment shows the activity of anti-insect pest, and described insect can comprise economically important agronomy insect, forestry pest, greenhouse insect, nursery pest, ornamental article insect, food and fiber insect, the public and animal health insect, family expenses and commercial building insect, houseware insect and storing insect.Insect pest comprises and is selected from Coleoptera, Diptera, Hymenoptera (Hymenoptera), lepidopteran, Mallophaga (Mallophaga), Homoptera (Homoptera), Orthoptera (Orthoptera), Thysanoptera (Thysanoptera), Dermaptera (Dermaptera), Isoptera (Isoptera), Anoplura (Anoplura), Siphonaptera (Siphonaptera), Trichoptera (Trichoptera) etc., particularly lepidopterous insect.

The larva of Coleoptera and adult comprise that the weevil of long angle Curculionidae (Anthribidae), Bruchidae (Bruchidae) and Culculionidae (Curculionidae) (includes but not limited to: anthonomus grandis (Anthonomus grandis Boheman, boll weevil); Rice water weevil (Lissorhoptrus oryzophilus Kuschel, rice water weevil); Grain weevil (Sitophilus granarius Linnaeus, granary weevil); Rice weevil (S.oryzae Linnaeus, rice weevil); Trifolium leaf resembles (Hypera punctata Fabricius, clover leaf weevil); Sunflower Receptacle stem weevil (Cylindrocopturus adspersus LeConte, sunflower stem weevil); Red seed weevil (Smicronyx fulvus LeConte, red sunflower seed weevil); Grey seed weevil (S.sordidus LeConte, gray sunflower seed weevil); Corn weevil (Sphenophorus maidis Chittenden, maize billbug); Flea beetle, the cucumber of Chrysomelidae (Chrysomelidae) are chrysomelid, rootworm, chrysomelid, colorado potato beetles and leaf miner (include but not limited to: state of Colorado colorado potato bug (Leptinotarsa decemlineata Say, Colorado potato beetle); Zea mays root firefly chrysomelid (Diabrotica virgifera virgifera LeConte, western corn rootworm); Pasteur's root chrysomelid (D.barberi Smith & Lawrence, northern corn rootworm); Cucumber 11 asterophyllite first (D.undecimpunctata howardi Barber, southern corn rootworm); Corn flea beetle (Chaetocnema pulicaria Melsheimer, corn flea beetle); Cruciferae flea beetle (Phyllotreta cruciferae Goeze, corn flea beetle); Grape colaspsis (Colaspis brunnea Fabricius, grape colaspis); Black angle scotellaris (Oulema melanopus Linnaeus, cereal leaf beetle); Sunflower Receptacle chrysomelid (Zygogramma exclamationis Fabricius, sunflower beetle)); The beetle of Coccinellidae (Coccinellidae) (includes but not limited to: mexican bean ladybird (Epilachna varivestis Mulsant, Mexican bean beetle); The chafer of dung beetle section (Scarabaeidae) and other beetle (include but not limited to: Japanese beetle (Popillia japonica Newman, Japanese beetle); North round end rhinoceros cockchafer (Cyclocephala borealis Arrow, northern masked chafer, white grub); South round end rhinoceros cockchafer (C.immaculata Olivier, southern masked chafer, white grub); Europe cockchafer (Rhizotrogus majalis Razoumowsky, European chafer); Grub (Phyllophaga crinita Burmeister, white grub); Radix Dauci Sativae beetle (Ligyrus gibbosus De Geer, carrot beetle)); The Carpet of Dermestidae (Dermestidae); The nematode of Elateridae (Elateridae): pseudo-wireworm (Eleodes spp.), wireworm (Melanotus spp.), single leaf click beetle (Conoderus spp.), Limonius spp., cone tail click beetle (Agriotes spp.), Ctenicera spp., Aeolus spp.; The beetle of the bark beetle of Scolytidae (Scolytidae) and TRenebrionidae (Tenebrionidae).

Lepidopterous larva includes but not limited to mythimna separata, cutworm, looper and the heliothine of Noctuidae (Noctuidae): autumn mythimna separata (Spodoptera frugiperda JE Smith, fall armyworm); Beet armyworm (S.exigua H ü bner, beet armyworm); Prodenia litura (S.litura Fabhcius, tobacco cutworm, cluster caterpillar); Bud band noctuid (Mamestra configurata Walker, bertha armyworm); Lopper worm (M.brassicae Linnaeus, cabbage moth); Black cutworm (Agrotis ipsilon Hufnagel, black cutworm); Western cutworm (A.orthogonia Morrison, western cutworm); Particle noctuid (A.subterranea Fabricius, granulate cutworm), kapok worm (Alabama argillacea H ü bner, cotton leaf worm); Cabbage looper (Trichoplusia ni H ü bner, cabbage looper); Soybean noctuid (Pseudoplusia includens Walker, soybean looper); Anticarsia (Anticarsia gemmatalis H ü bner, velvetbean caterpillar); The green noctuid of clover (Hypena scabra Fabricius, green cloverworm); Heliothis virescens (Heliothis virescens Fabricius, tobacco budworm); One star mythimna separata (Pseudaletia unipuncta Haworth, armyworm); Tertia noctuid (Athetis mindara Barnes and Mcdunnough, rough skinned cutworm); Dark edge cutworm (Euxoa messoria Harris, darksided cutworm); Egyptian golden steel bores (Earias insulana Boisduval, spiny bollworm); Emerald green line gold steel bores (E.vittella Fabricius, spotted bollworm); Heliothis zea (Helicoverpa armigera H ü bner, American bollworm); The real noctuid (H.zea Boddie, corn earworm or cotton bollworm) of paddy; Line butterfly caterpillar (Melanchra picta Harris, zebra caterpillar); Oranges and tangerines noctuid (Egira (Xylomyges) curialis Grote, citrus cutworm); The boring worm of Pyralidae (Pyralidae), casebearer, web spinner, cone moth and spot moth: European corn borer (Ostrinia nubilalis H ü bner, European corn borer); Navel orangeworm (Amyelois transitella Walker, naval orangeworm); Anagasta kuehniella (Anagasta kuehniella Zeller, Mediterranean flour moth); Cadra cautella (Cadra cautella Walker, almond moth); Striped rice borer (Chilo suppressalis Walker, rice stem borer); Spot dogstail snout moth's larva (C.partellus, sorghum borer); Rice moth (Corcyra cephalonica Stainton, rice moth); Zea mays root web spinner (Crambus caliginosellus Clemens, corn root webworm); Bluegrass web spinner (C.teterrellus Zincken, bluegrass webworm); Cnaphalocrocis medinalls guenee (Cnaphalocrocis medinalis Guenee, rice leaf roller); Grape leaf folder (Desmia funeralis H ü bner, grape leaffolder); Melon worm (Diaphania hyalinata Linnaeus, melon worm); Pickles worm (D.nitidalis Stoll, pickleworm); Southwest Maize snout moth's larva (Diatraea grandiosella Dyar, southwestern corn borer); Little sugarcane borer (D.saccharalis Fabricius, surgarcane borer); Mexico's rice borer (Eoreuma loftini Dyar, Mexican rice borer); Cacac moth (Ephestia elutella H ü bner, tobacco (cacao) moth); Galleria mellonella waxmoth (Galleria mellonella Linnaeus, greater wax moth); Wild snout moth's larva (Herpetogramma licarsisalis Walker, sod webworm); Sunflower Receptacle phycitid (Homoeosoma electellum Hulst, sunflower moth); South America maize seedling phycitid (Elasmopalpus lignosellus Zeller, lesser cornstalk borer); Lesser wax-moth (Achroia grisella Fabricius, lesser wax moth); Loxostege sticticalis (Loxostege sticticalis Linnaeus, beet webworm); Tea tree snout moth's larva (Orthaga thyrisalis Walker, tea tree web moth); The wild snout moth's larva (Maruca testulalis Geyer, bean pod borer) of beans; Indian meal moth (Plodia interpunctella H ü bner, Indian meal moth); Yellow rice borer (Scirpophaga incertulas Walker, yellow stem borer); Greenhouse snout moth's larva (Udea rubigalis Guenee, celery leaftier); And the tortrix moth of Tortricidae (Tortricidae), aphid, the real worm of kind and fruit worm: blackhead leaf roller (Acleris gloverana Walsingham, Western blackheaded budworm); Blackhead Acleris spp (A.variana Fernald, Eastern blackheaded budworm); The yellow volume of fruit tree moth (Archips argyrospila Walker, fruit tree leaf roller); Europe leaf roller (A.rosana Linnaeus, European leaf roller); And other Archips spp (Archips) species: adoxophyes moth (Adoxophyes orana Fischer von summer fruittortrix moth); Striped sunflower moth (Cochylis hospes Walsingham, banded sunflower moth); Hazel steinernema (Cydia latiferreana Walsingham, filbertworm); Carpocapsa pononella (C.pomonella Linnaeus, coding moth); Variegated leaf roller (Platynota flavedana Clemens, variegated leafroller); Carnation steinernema (P.stultana Walsingham, omnivorous leafroller); Vitis vinifera olethreutid (Lobesia botrana Denis & Schifferm ü ller, European grape vine moth); Spilonota lechriaspis (Spilonota ocellana Denis & Schifferm ü ller, eyespotted bud moth); Grape fruit moth (Endopiza viteana Clemens, grape berry moth); Ligustrum fine tortricidae (Eupoecilia ambiguella H ü bner, vine moth); Brazil apple skin worm (Bonagota salubricola Meyrick, Brazilian apple leafroller); Oriental fruit months (Grapholita molesta Busck, oriental fruit moth); Sunflower Receptacle bud moth (Suleima helianthana Riley, sunflower budmoth); Silver lap moth (Argyrotaenia spp.); A tail a kind of butterfly harmful to crop plants (Choristoneura spp.).

Lepidopterous selected other agronomy insect includes but not limited to: fall cankerworm (Alsophila pometaria Harris, fall cankerworm); Peach branch gelechiid (Anarsia lineatella Zeller, peach twig borer); Rhinoceros volume moth (Anisota senatoria J.E.Smith, orange striped oakworm); Tussah (Antheraea pernyi Guerin-Meneville, Chinese Oak Silkmoth); Silkworm (Bombyx mori Linnaeus, Silkworm); Cotton lyonetid (Bucculatrixthurberiella Busck, cotton leaf perforator); The yellow butterfly (Colias eurytheme Boisduval, alfalfa caterpillar) of clover; The yellow butterfly (Datana integerrima Grote & Robinson, walnut caterpillar) of English walnut; Dendrolimus sibiricus (Dendrolimus sibiricus Tschetwerikov, Siberian silk moth); White looper (Ennomos subsignariaH ü bner, elm spanworm); Bodhi looper (Erannis tiliaria Harris, linden looper); Pornography and drug moth (Euproctis chrysorrhoea Linnaeus, browntail moth); Black plan sandfly moth (Harrisina americana Guerin-Meneville, grapeleaf skeletonizer); Scope caterpillar moth (Hemileuca oliviae Cockrell, range caterpillar); Fall webworms (Hyphantria cunea Drury, fall webworm); The moth-eaten moth (Keiferia lycopersicella Walsingham, tomato pinworm) of tomato; Polyura narcaea (Lambdina fiscellariafiscellaria Hulst, Eastern hemlock looper); Western hemlock looper (L.fiscellaria lugubrosa Hulst, Western hemlock looper); Leucoma candida (Leucoma salicis Linnaeus, satin moth); Gypsymoth (Lymantria dispar Linnaeus, gypsy moth); Tomato hawkmoth (Manduca quinquemaculata Haworth, five spotted hawk moth, tomato homworm); Tobacco sky (M.sexta Haworth, tomato hornworm, tobacco hornworm); Winter looper (Operophtera brumata Linnaeus, winter moth); Spring looper (Paleacrita vernata Peck, spring cankerworm); Large swallowtail butterfly (Papilio cresphontes Cramer, giant swallowtail, orange dog); California strain worm (Phryganidia californica Packard, California oakworm); Phyllocnistis citrella stainton (Phyllocnistis citrella Stainton, citrus leafminer); Spot curtain leaf miner (Phyllonorycter blancardella Fabricius, spotted tentiform leafminer); Large white butterfly (Pieris brassicae Linnaeus, large white butterfly); Pieris rapae (P.rapae Linnaeus, small white butterfly); Dark arteries and veins small white (P.napi Linnaeus, green veined white butterfly); Arithoke plume moth (Platyptilia carduidactyla Riley, artichoke plume moth); Water chestnut spot is starved (Plutella xylostella Linnaeus, diamondback moth); Pink bollworm (Pectinophora gossypiella Saunders, pink bollworm); South diamond-back moth (Pontia protodice Boisduval & Leconte, Southern cabbageworm); Omnivorous looper (Sabulodes aegrotata Guenee, omnivorous looper); Blatta concinna (Schizura concinna J.E.Smith, red humped caterpillar); Gelechiid (Sitotroga cerealella Olivier, Angoumois grain moth); Song Yi is with moth (Thaumetopoea pityocampa Schiffermuller, pine processionary caterpillar); The casemaking clothes moth (Tineola bisselliella Hummel, webbing clothesmoth) of knotting; Liriomyza brponiae (Tuta absoluta Meyrick, tomato leafminer); Apple ermine moth (Yponomeuta padella Linnaeus, ermine moth); Heliothis subflexa Guenee; Tent caterpillar (Malacosoma spp); And poison moth (Orgyia spp).

Dipterous adult and larva comprise: such as the Liriomyza of corn spot Liriomyza (Agromyza parvicornis Loew, corn blotch leafminer); Mosquito (includes but not limited to: Chinese sorghum cecidomyiia (Contarinia sorghicola Coquillett, sorghum midge); Hessian fly (Mayetiola destructor Say, Hessian fly); Wheat midge (Sitodiplosis mosellana Gehin, wheat midge); Sunflower seeds mosquito (Neolasioptera murifeldtiana Felt, sunflower seed midge)); Fruit fly (Tephritidae (Tephritidae)), Sweden stem maggot (Oscinella frit Linnaeus, frit flies); Maggot (includes but not limited to: delia platura (Delia platura Meigen, seedcorn maggot); Wheat bulb fly (D.coarctata Fallen, wheat bulb fly); And fly (Delia spp.), America stem maggot (Meromyza americana Fitch, wheat stem maggot) are planted in other ground; House fly (Musca domestica Linnaeus, house flies); Fannia canicularis (Fannia canicularis Linnaeus), hutch fly (F.femoralis Stein, lesser house flies); Tatukira (Stomoxys calcitrans Linnaeus, stable flies)); Face fly, horn fly, calliphorid, golden fly (Chrysomya spp.); Volt fly (Phormia spp.); And other liver moss fly insect, horse botfly, horsefly (Tabanus spp.); Stomach fly, stomach fly (Gastrophilus spp.); Botfly (Oestrus spp.); Bomb fly, torsalo (Hypoderma spp.); Spot horsefly, spot horsefly (Chrysops spp.); Sheep hippoboscid (Melophagus ovinus Linnaeus, keds); And other Brachycera (Brachycera), mosquito, yellow-fever mosquito (Aedes spp.); Anopheles (Anopheles spp.); Culex (Culex spp.); Black fly, former buffalo gnat (Prosimulium spp.); Buffalo gnat (Simulium spp.); Sting midge, sand fly, mushroom fly and other Nemoceras (Nematocera).

Adult and the nymph of Hemiptera and Homoptera comprise insect, such as, but not limited to: the leafhopper of the fleahopper of the adelgid of Adelgidae (Adelgidae), Miridae (Miridae), the cicada of Cicadidae (Cicadidae), Cicadellidae (Cicadellidae), smaller green leaf hopper (Empoasca spp.); The plant hopper of water chestnut Delphacidae (Cixiidae), Flatidae (Flatidae), fulgoroidea (Fulgoroidea), circle Delphacidae (Issidae) and Dao Shi section (Delphacidae); The horned frog of Membracidae (Membracidae); The wood louse of Psyllidae (Psyllidae); The aleyrodid of Aleyrodidae (Aleyrodidae); The aphid of Aphidiadae (Aphididae); The Phylloxera of Phylloxera Aphididae (Phylloxeridae); The mealybug of Pseudococcidae (Pseudococcidae); The scale insect of chain Coccidae (Asterolecanidae), soft Coccidae (Coccidae), fuchsin a red-spotted lizard section (Dactylopiidae), shield Coccidae (Diaspididae), Eriococcinae (Eriococcidae), ancient type of banner hoisted on a featherdecked mast Coccidae (Ortheziidae), thorn certain herbaceous plants with big flowers Coccidae (Phoenicococcidae) and large Coccidae (Margarodidae); The lace bug of Tingidae (Tingidae); The Stink Bug of Pentatomiddae (Pentatomidae); The China bug of Lygaeidae (Lygaeidae), long Chinese toon (Blissus spp.); And other stinkbug class (seed bugs), the squash bug of the froghopper of Cercopidae (Cercopidae), Coreidae (Coreidae) and tetranychus autumnalis and the cotton stinkbug of Pyrrhocoridae (Pyrrhocoridae).

In Homoptera, on agronomy, important member also includes but not limited to: acyrthosiphum pisim (Acyrthisiphon pisum Harris, pea aphid); Cowpea aphid (Aphis craccivora Koch, cowpea aphid); Black bean aphid (A.fabae Scopoli, black bean aphid); Cotten aphid (A.gossypii Glover, cotton aphid, melon aphid); Corn root aphid (A.maidiradicis Forbes, corn root aphid); Apple yellow aphid (A.pomi De Geer, apple aphid); Spiraea aphid (A.spiraecola Patch, spirea aphid); Eggplant ditch is without net aphid (Aulacorthum solani Kaltenbach, foxglove aphid); Strawberry nail aphid (Chaetosiphon fragaefolii Cockerell, strawberry aphid); The two tail aphids (Diuraphis noxia Kurdjumov/Mordvilko, Russian wheat aphid) of wheat; Rose apple aphid (Dysaphis plantaginea Paaserini, rosy apple aphid); Eriosoma lanigerum (Eriosoma lanigerum Hausmann, woolly apple aphid); Brevicoryne brassicae (Brevicoryne brassicae Linnaeus, cabbage aphid); Hyaloptera aphid (Hyalopterus pruni Geoffroy, mealy plum aphid); Radish aphid (Lipaphis erysimi Kaltenbach, turnip aphid); Acyrthosiphon dirhodum (Metopolophium dirrhodum Walker, cereal aphid); Root of Beijing euphorbia Macrosiphus spp (Macrosiphum euphorbiae Thomas, potato aphid); Cigarette black peach aphid (Myzus persicae Sulzer, peach-potato aphid, green peach aphid); Lettuce aphid (Nasonovia ribisnigri Mosley, lettuce aphid); Goitre woolly aphid (Pemphigus spp., root aphids and gall aphids); Corn leaf aphids (Rhopalosiphum maidis Fitch, corn leaf aphid); Rhopalosiphum padi (R.padi Linnaeus, bird cherry-oat aphid); Green bugs (Schizaphis graminum Rondani, greenbug); Yellow sugarcane aphid (Sipha flava Forbes, yellow sugarcane aphid); Grain aphid (Sitobion avenae Fabricius, English grain aphid); Clover spot aphid (Therioaphis maculata Buckton, spotted alfalfa aphid); Black oranges and tangerines aphid (Toxoptera aurantii Boyer de Fonscolombe, black citrus aphid) and brown citrus aphid (T.citricida Kirkaldy, brown citrus aphid); Adelgid (Adelges spp., adelgids); Pecan Phylloxera (Phylloxera devastatrix Pergande, pecan phylloxera); Sweet potato whitefly (Bemisia tabaci Gennadius, tobacco whitefly, sweetpotato whitefly); Bemisia argentifolii (B.argentifolii Bellows & Perring, silverleaf whitefly); Citrus whitefly (Dialeurodes citri Ashmead, citrus whitefly); Bemisia tabaci (Trialeurodes abutiloneus, bandedwinged whitefly) and Trialeurodes vaporariorum Westwood (T.vaporariorum Westwood, greenhouse whitefly); Potato smaller green leaf hopper (Empoasca fabae Harris, potato leafhopper); Small brown rice planthopper (Laodelphax striatellus Fallen, smaller brown planthopper); Aster leafhopper (Macrolestes quadrilineatus Forbes, aster leafhopper); Rice green leafhopper (Nephotettix cinticeps Uhler, green leafhopper); Article two, rice green leafhopper (N.nigropictus Stal, rice leafhopper); Brown paddy plant hopper (Nilaparvata lugens Stal, brown planthopper); Corn plant hopper (Peregrinus maidis Ashmead, corn planthopper); White backed planthopper (Sogatella furcifera Horvath, white-backed planthopper); Planthopper (Sogatodes orizicola Muir, rice delphacid); The white leafhopper of apple (Typhlocyba pomaria McAtee, white apple leafhopper); Grape leafhopper (Erythroneoura spp., grape leafhoppers); 17 years cicada (Magicicada septendecim Linnaeus, periodical cicada); Icerya purchasi (Icerya purchasi Maskell, cottony cushion scale); Theatre armored scale (Quadraspidiotus perniciosus Comstock, San Jose scale); Planococus cirri (Planococcus citri Risso, citrus mealybug); The kind of mealybug (Pseudococcus spp.) (other the group of mealybug system); Pear sucker (Cacopsylla pyricola Foerster, pear psylla); Kaki lice (Trioza diospyri Ashmead, persimmon psylla).

In Hemiptera, on agronomy, important species include but not limited to: Nezara viridula smaragdula Fabricius. (Acrosternum hilare Say, green stink bug); Squash bug (Anasa tristis De Geer, squash bug); China bug (Blissus leucopterus leucopterus Say, chinch bug); Side wing lace bug (Corythuca gossypii Fabricius, cotton lace bug); Tomato stinkbug (Cyrtopeltis modesta Distant, tomato bug); Cotton stinkbug (Dysdercus suturellus Herrich-Schaffer, cotton stainer); Brown smelly stinkbug (Euschistus servus Say, brown stink bug); The smelly stinkbug of one spot (E.variolarius Palisot de Beauvois, one-spotted stink bug); Chinch bug (Graptostethus spp.) (the fruit stinkbug group of system (complex of seed bugs)); Pine needle root stinkbug (Leptoglossus corculus Say, leaf-footed pine seed bug); U.S. tarnished plant bug (Lygus lineolaris Palisot de Beauvois, tarnished plant bug); Beanpod lygus bug (L.Hesperus Knight, Western tarnished plant bug); Tarnished plant bug (L.pratensis Linnaeus, common meadow bug); Lygus bug (L.rugulipennis Poppius, European tarnished plant bug) becomes mildewed; Long green plant bug (Lygocoris pabulinus Linnaeus, common green capsid); Paddy rice Nezara viridula smaragdula Fabricius. (Nezara viridula Linnaeus, southern green stink bug); Rice stinkbug (Oebalus pugnax Fabricius, rice stink bug); Coign chinch bug (Oncopeltus fasciatus Dallas, large milkweed bug); Cotton plant bug (Pseudatomoscelis seriatus Reuter, cotton fleahopper).

The insect that Hemiptera comprises comprises: strawberry stinkbug (Calocoris norvegicus Gmelin, strawberry bug); Orthops campestris Linnaeus; Apple capsid (Plesiocoris rugicollis Fallen, apple capsid); Tomato stinkbug (Cyrtopeltis modestus Distant, tomato bug); The little fleahopper of tobacco (Cyrtopeltis notatus Distant, suckfly); Hickie fleahopper (Spanagonicus albofasciatus Reuter, whitemarked fleahopper); Chinese honey locust stinkbug (Diaphnocoris chlorionis Say, honeylocust plant bug); Onion stinkbug (Labopidicola allii Knight, onion plant bug); Cotton plant bug (Pseudatomoscelis seriatus Reuter, cotton fleahopper); Rapid plant bug (Adelphocoris rapidus Say, rapid plant bug); Four line fleahoppers (Poecilocapsus lineatus Fabricius, four-lined plant bug); Intend China bug (Nysius ericae Schilling, false chinch bug); Tea golden thistle horse (Nysius raphanus Howard, false chinch bug); The blue or green stinkbug (Nezara viridula Linnaeus, Southern green stink bug) of rice; Eurygasterspp (Eurygaster spp.); Coried (Coreidae spp.); Red stinkbug (Pyrrhocoridae spp.); Rain moth (Tinidae spp.); Belostomatid (Blostomatidae spp.); Hunt stinkbug (Reduviidae spp.); And smelly stinkbug (Cimicidae spp.).

Mite (Acari) (mite (mites)) object adult and larva comprise: wheat leaf roll mite (Aceriatosichella Keifer, wheat curl mite); The little Acarus hordei of brown (Petrobia latens M ü ller, brown wheat mite); Spider mite and the trombiculid of Tetranychidae (Tetranychidae), European tetranychid (Panonychus ulmi Koch, European red mite); Tetranychus urticae (Tetranychus urticae Koch, two spotted spider mite); Step tetranychid (T.mcdanieli McGregor, McDaniel mite); Carmine spider mite (T.cinnabarinus Boisduval, carmine spider mite); Turkestan tetranychid (T.turkestani Ugarov & Nikolski, strawberry spider mite); The flat mite of Tenuipalpidae (Tenuipalpidae), grape brevipalpus (Brevipalpus lewisi McGregor, citrus flat mite); The rust mite of Eriophyidae (Eriophyidae) is with Ya Ying mite and other food tetranychid and to the healthy important mite of human and animal, rust mite in Ji Qing section (Epidermoptidae), demodex folliculorum in Demodicidae (Demodicidae), the paddy mite of Shi Tian mite section (Glycyphagidae), the flat lice of Ying Pi section (Ixodidae), Blacklegged tick (Ixodes scapularis Say, deer tick); Ixodes holocyclus (I.holocyclus Neumann, Australian paralysis tick); U.S.'s dog tick (Dermacentor variabilis Say, American dog tick); America tick (Amblyomma americanum Linnaeus, lone star tick); And itch mite and the itch mite of itch mite section (Psoroptidae), Pyemotidae (Pyemotidae) and Sarcoptidae (Sarcoptidae).

Insect pest in Thysanura (Thysanura) comprises: silverfiss (Lepisma saccharina Linnaeus, silverfish); Special mess silverfish (Thermobia domestica Packard, firebrat).Other arthropod pests comprises the spider in Araneida (Araneae), as brown reclusion spider (Loxosceles reclusa Gertsch & Mulaik, brown recluse spider); And latrodectus mactans (Latrodectus mactans Fabricius, black widow spider); With the centipede in common house centipede order (Scutigeromorpha), as common house centipede (Scutigera coleoptrata Linnaeus, house centipede).

Can grow in early days, for example, as larva or other prematurity form, the insecticidal activity of the composition of test implementation mode to insect pest.Can, under complete darkness condition, in approximately 20 ℃ to approximately 30 ℃, approximately 30%, to approximately 70% relative humidity, cultivate insect.Can be as Czaplaand Lang (1990) J.Econ.Entomol.83 (6): as described in 2480-2485, carry out bioanalysis.Cultivation insect larvae as well known to those skilled in the art and the method for carrying out bioanalysis.

The known various bioassay techniques of those skilled in the art.Common program comprises to the diet source of sealing in container adds experimental compound or organism.Can through but not limited to take food and expose that mortality ratio after suitably long-time changes, loses weight, attracts, driving property and other behavior and physical change, measure insecticidal activity.Can take food insect pest to larval stage or adult stage any and use bioanalysis as herein described.

By explanation, unrestriced mode is stated the following example.

Embodiment

Embodiment 1: the bioanalysis of test bacillus thuringiensis toxin to the insecticidal activity of selected insect

Carry out bioanalysis and kill the effect of insect toxins peptide to west corn rootworm with the Bt evaluating as described in SEQ ID NO:2.The artificial diet that comprises insecticidal protein is taken food to analysis.With the artificial diet of Coleoptera specificity, carry out topical application insecticidal protein.With the ratio application toxin of the every 25 μ l sample 1.0 μ g of every container, and be dried.This albumen is included in the 10mM carbonate buffer solution of pH 10.Each container is placed a newborn larvae, arbitrarily to take food 5 days.If have such as atrophy and or dead larva reaction, result is positive.If larva is similar to the negative control that eats the diet of only applying above-mentioned damping fluid, result is negative.

Table 1:SEQ ID NO:2 takes food bioanalysis result

Embodiment 2: determine LC ₅₀and EC ₅₀

Carry out bioanalysis and as described in SEQ ID NO:2, kill the LC of insect toxins peptide to west corn rootworm (Zea mays root firefly is chrysomelid) to determine ₅₀and EC ₅₀.The artificial diet that comprises insecticidal protein is taken food to analysis.The 10mM carbonate buffer solution of pH 10 and the dilution of insect diet for insecticidal protein, to reach 50000,5000,500,50 and the toxin final concentration of 5ppm.Each container is placed a newborn larvae, arbitrarily to take food 5 days.Under each dosage, octuplicately carry out each bioanalysis, by described bioanalysis in triplicate.By mortality ratio, result is shown and made LC ₅₀and/or shown and made EC by weighing survival larva under each toxin concentration ₅₀.

Embodiment 3: by particle bombardment and regeneration of transgenic Plant Transformation corn

For example, with (containing toxin nucleotide sequence, SEQ ID NO:1) DNA molecular bombardment is from the prematurity maize of greenhouse donor plant, described toxin nucleotide sequence and ubiquitin promoter and selectable marker gene PAT (Wohlleben et al. (1988) Gene 70:25-37) are operatively connected, and it gives the resistance to two the third ammonia phosphorus weedicides.Selectively, on independent DNA molecular, provide and can select marker gene.Transform as follows.Culture medium prescription is as follows.

the preparation of target tissue

To fringe peeling, use 30%CLOROX ^tMsYNTHETIC OPTICAL WHITNER adds 0.5% micro-washing agent by its surface disinfection 20 minutes, with twice of distilled water rinsing.Cut and place immature embryo, under plumular axis one side direction (in cotyledon dish one side direction), 25 embryos of every plate, place 4 hours in 560Y substratum, subsequently it are arranged in the target region of 2.5cm, in order to bombardment.

the preparation of DNA

The plasmid vector that preparation comprises the toxin nucleotide sequence (for example SEQID NO:1) being operatively connected with ubiquitin promoter.For example, suitable conversion carrier comprises and the corn UBI1 promotor of PinII terminator combination, 5 ' UTR and the UBI1 intron of UBI1.The PAT that this carrier comprises CAMV35S promoters driven in addition can select marker gene, and comprises CAMV35S terminator.Optionally, can select mark can be positioned on independent plasmid.Use following CaCl2 precipitation program, by comprising toxin nucleotide sequence and PAT, can select the DNA molecular of mark to be precipitated to 1.1 μ m (mean diameter) tungsten balls:

100 μ L are containing the water of prepared tungsten particle

10 μ L (1 μ g) are dissolved in the DNA (the total DNA of 1 μ g) of Tris edta buffer liquid

100μL?2.5M?CaCl ₂

10 μ L 0.1M spermidines

Each reagent is added to tungsten particle suspension in turn, be placed on multitube vortice simultaneously.By the of short duration ultrasonication of final mixture, and it is hatched 10 minutes under lasting vortex.At precipitation after date, of short duration centrifugal described pipe, removes liquid, cleans centrifugal 30 seconds with 500mL 100% ethanol.Remove again liquid, 105 μ L 100% ethanol are added into final tungsten particle bead.For particle gun bombardment, by the of short duration supersound process of this tungsten/DNA grain, and by 10 μ L points to each larger vector (macrocarrier) center, and make it be dried approximately 2 minutes, bombard subsequently.

particle gun is processed

With particle gun #HE34-1 or #HE34-2, under horizontal #4, bombard sample panel.All samples is accepted the single shooting under 650PSI, and altogether in decuplicate, it takes from each pipe with prepared grain/DNA.

processing subsequently

After bombardment, embryo is placed in to 560Y substratum 2 days, is gone to subsequently and contain the 560R selection substratum that 3mg/ rises two the third ammonia phosphorus, the cultivation of going down to posterity in every 2 weeks.Select after approximately 10 weeks, the callus clone of anti-selection is gone to 288J substratum, with initial plant regeneration.After somatic embryo ripe (2-4 week), well-developed somatic embryo is gone to the substratum that sprouts, and go to illumination cultivation chamber.After about 7-10 days, developmental seedling is gone to 272V in pipe without hormone culture-medium, cultivate 7-10 days, until seedling is well built up.Subsequently plant is gone on the mat of the tray that contains potting soil (be equal to 2.5 " basin); make it growth indoor growing 1 week; in greenhouse, to grow 1-2 week more subsequently, then transfer them to typical 600 basins (1.6 gallons), and make it grow to maturation.By analysis known in the art or as above, monitor and the toxin of the plant of marking is expressed.

bombardment and the substratum of cultivating

Bombardment substratum (560Y) comprises the basic salt of 4.0g/L N6 (SIGMA C-1416), 1.0mL/L Eriksson vitamine mixture (1000x SIGMA-1511), 0.5mg/L thiamines HCl, 120.0g/L sucrose, 1.0mg/L 2,4-D and 2.88g/L L-PROLINE (are adjusted to after pH5.8 with KOH, use dl H ₂o constant volume); 2.0g/L Gelrite ^tM(use dl H ₂after O constant volume, add); With 8.5mg/L Silver Nitrate (adding by medium sterilization and after being cooled to room temperature).Select substratum (560R) to comprise the basic salt of 4.0g/L N6 (SIGMA C-1416), 1.0mL/L Eriksson vitamine mixture (1000x SIGMA-1511), 0.5mg/L thiamines HCl, 30.0g/L sucrose, with 2.0mg/L 2,4-D (is adjusted to after pH 5.8 with KOH, uses dl H ₂o constant volume); 3.0g/LGelrite ^tM(use dl H ₂after O constant volume, add); With 0.85mg/L Silver Nitrate and two the third ammonia phosphorus (all adding by medium sterilization and after being cooled to room temperature) of 3.0mg/L.

Plant regeneration substratum (288J) comprises 4.3g/L MS salt (GIBCO 11117-074), and (0.10g/L pyridoxol HCl and 0.40g/L glycine, with refining D-I H for 0.100g nicotinic acid, 0.02g/L thiamines HCl for 5.0mL/L MS VITAMIN mother liquor ₂o constant volume) (Murashige and Skoog (1962) Physiol.Plant.15:473), 100mg/L myo-inositol, 0.5mg/L zeatin, 60g/L sucrose, and the 0.1mM dormin of 1.0mL/L (is adjusted to after pH 5.6, with refining dI H ₂o constant volume); 3.0g/L Gelrite ^tM(use dl H ₂after O constant volume, add); With 1.0mg/L indolylacetic acid and two the third ammonia phosphorus of 3.0mg/L (all adding by medium sterilization and after being cooled to 60 ℃).

Without hormone culture-medium (272V), comprise 4.3g/L MS salt (GIBCO 11117-074), (0.10g/L pyridoxol HCl, and 0.40g/L glycine, with refining dl H for 0.100g nicotinic acid, 0.02g/L thiamines HCl for 5.0 mL/L MS VITAMIN mother liquors ₂o constant volume), 0.1g/L myo-inositol, and 40.0g/L sucrose (is adjusted after pH to 5.6, with refining dl H ₂o constant volume); (use refining dl H with 6g/L bacterial agar ₂after O constant volume, add), by its sterilizing and be cooled to 60 ℃.

Embodiment 4: agriculture bacillus mediated corn transforms and transgenic plant regeneration

For for example, agriculture bacillus mediated corn with toxin nucleotide sequence (, SEQ ID NO:1), transform, can use method (United States Patent (USP) the 5th, 981, No. 840 and the open WO98/32326 of PCT patent of Zhao; Its content is incorporated to herein by reference).In brief, from corn dividing from immature embryo, this embryo is contacted with agrobacterium suspension, and described contact can be transferred to toxin nucleotide sequence (SEQ ID NO:1) on described bacterium under the condition of at least one cell of at least one immature embryo carries out (step 1: infect step).In this step, immature embryo can be immersed to agrobacterium suspension with initial inoculation.Embryo and Agrobacterium are cultivated to certain hour (step 2: be total to culturing step) altogether.After infecting step, immature embryo is cultivated on solid medium.After this common incubation period, relate to optional " tranquillization " step.In this tranquillization step, having the microbiotic of at least one known inhibition Agrobacterium growth but not adding under the selective reagents for vegetable transformant, hatch described embryo (step 3: tranquillization step).For removing Agrobacterium and being the quiescent stage of institute's cells infected, immature embryo is had to microbiotic but cultivating on solid medium without selective reagents.Next, inoculated embryo is cultivated containing on the substratum of selective reagent, and the transformed calli in growth is recovered to (step 4: select step).Immature embryo is cultivated having on the solid medium of selective reagent, caused the selective growth of institute's transformant.Make subsequently callus regeneration become plant (step 5: regeneration step), the callus of growing is cultivated on solid medium, with aftergrowth on selective medium.

Embodiment 5: soybean transformation embryo

The following plasmid bombardment soybean embryo with containing the SEQ ID NO:1 toxin nucleotide sequence being operatively connected with pinII promotor.For inductor somatic embryo, the cotyledon of the long 3-5mm that the immature seed of the surface disinfection from suitable soybean culture kind is cut is cultivated 6-10 week in 26 ℃ at suitable nutrient agar illumination or dark.Cut subsequently the somatic embryo that produces secondary embryo, and inserted in suitable liquid nutrient medium.Repeat to select as the somatic embryo cell mass of early stage, globular stage embryo multiplication after, maintain as follows described suspension.

In 35mL liquid nutrient medium by soybean embryo generation suspension culture on 150rpm rotation rocker, maintain in 26 ℃, described in maintain 16:8 hour daytime/night timetable fluorescence under carry out.By the tissue of about 35mg is seeded to 35mL liquid nutrient medium, the every two weeks cultivation cultures that go down to posterity.

By particle gun, bombard method (Klein et al. (1987) Nature (London) 327:70-73, United States Patent (USP) the 4th, 945, No. 050), soybean transformation embryo generation suspension culture subsequently.Can be by the particle gun PDS1000/HE of Du Pont equipment (helium installs additional) for these conversions.

Can be by following formed transgenosis for the marker gene selected of promotion transformation of soybean: cauliflower mosaic virus 35 S promoter (Odell et al. (1985) Nature 313:810-812), plasmid pJR225 are (from intestinal bacteria; Gritz et al. (1983) Gene 25:179-188) hygromycin phosphotransferase gene and the nopaline synthase gene 3rd ' district of agrobacterium tumefaciens Ti-plasmids T-DNA.Can the expression cassette that comprise the toxin nucleotide sequence (for example, SEQ ID NO:1) being operatively connected with pinII promotor is separated as restriction fragment.This fragment can be inserted to unique restriction site of the carrier that carries marker gene subsequently.

60mg/mL 1 μ m goldc grains suspension (successively) to 50 μ L adds 5 μ L DNA (1 μ g/ μ L), 20 μ L spermidines (0.1M) and 50 μ L CaCl ₂(2.5M).By this prepared product jolting 3 minutes, in microcentrifuge, rotate 10 seconds subsequently, supernatant liquor is removed.With 400 μ L 70% ethanol, the coated particle of DNA is cleaned once subsequently, and it is resuspended in the dehydrated alcohol of 40 μ L.Can be by DNA/ grain suspension ultrasonication 3 times, each 1 second.Subsequently by the coated goldc grains loading of 5 microlitre DNA to each larger vector dish.

The suspension culture in two week age of about 300-400mg is placed in to empty 60 * 15mm Petri dish, and with pipettor, residual liquid is removed from tissue.To each transformation experiment, conventionally bombard the tissue of an about 5-10 plate.Film destroy pressure is located to 1100psi, this chamber is evacuated to 28 inches of mercury vacuum.Placing tissue at a distance from approximately 3.5 inches of retaining screens, bombarded three times.After bombardment, tissue is cut in half, and put back to liquid, and cultivate as mentioned above.

5-7 days after bombardment, can change liquid nutrient medium with fresh culture, and 11-12 days after bombardment can change with the fresh culture that contains 50mg/mL Totomycin.Can upgrade weekly this selective medium.In 7-8 week after bombardment, can observe from unconverted downright bad cells,primordial and roll into a ball the green transforming tissue of growth.Can remove separated chlorenchyma, and be seeded to single flask,, the embryo generation suspension culture that transform new, clonal expansion to generate.Can process each new system as transformation event independently.Can go down to posterity subsequently and cultivate these suspension, and the cell mass using it as immature embryo maintains, or by the ripe of individual somatic embryo with sprout, by the whole plant of its regeneration.

All publications, patent and the patent application that present specification is mentioned represents those skilled in the art of the invention's level.By reference all publications, patent and patent application are incorporated to herein, to reach as each independent publication, patent or patent application, specifically and are individually indicated as being the degree being incorporated to by reference.

Although be the clear object of understanding, by explanation and example, in certain details, invention has above been described, clearly can in the scope of embodiment, put into practice some variation and modification.

Claims (25)

1. the nucleic acid molecule of separation, is selected from:

A) nucleic acid molecule being formed by SEQ ID NO:1 nucleotide sequence; With

The nucleic acid molecule of the polypeptide that b) coding SEQ ID NO:2 aminoacid sequence forms.

2. the nucleic acid molecule of separation as claimed in claim 1, wherein said nucleotide sequence is the composition sequence that is designed to express in plant.

3.DNA construct, comprises nucleic acid molecule as claimed in claim 1.

4. DNA construct as claimed in claim 3, also comprises the nucleic acid molecule of coding allos mark.

5. produce the method for host cell, it comprises introduces described host cell by DNA construct claimed in claim 3.

6. method as claimed in claim 5, described cell is bacterial cell.

7. method as claimed in claim 5, described cell is vegetable cell.

8. produce the method for transgenic plant, it comprises makes in described plant, to have host cell claimed in claim 7.

9. method as claimed in claim 8, wherein said plant is selected from corn, jowar, wheat, Sunflower Receptacle, tomato, cress, green pepper, potato, cotton, paddy rice, soybean, beet, sugarcane, tobacco and barley.

10. method as claimed in claim 9, wherein said cress is Caulis et Folium Brassicae capitatae or rape.

11. Accessory Right require the method for the seed of the plant generation conversion that described in 9, method produces, and wherein said seed comprises described DNA construct.

12. have the isolated polypeptide of insecticidal activity, and it is by the nucleotide sequence coded polypeptide of SEQ ID NO:1.

13. polypeptide as claimed in claim 12, itself and allos mark sequence merge.

14. compositions, comprise polypeptide as claimed in claim 12.

15. compositions as claimed in claim 14, wherein said composition is selected from powder, pulvis, bead, particle, sprays, emulsion, colloid and solution.

16. compositions as claimed in claim 14, wherein prepare described composition by dry, homogenate, extraction, filtration, centrifugal, sedimentation or concentrated bacillus thuringiensis (Bacillus thuringiensis) cell culture.

17. compositions as claimed in claim 16, wherein prepare described composition by freeze-drying bacillus thuringiensis (Bacillus thuringiensis) cell culture.

18. compositions as claimed in claim 14, comprise 1% to 99% described polypeptide by weight.

19. control the method for coleopteran pest colony, comprise described colony is contacted with the polypeptide as claimed in claim 12 of insecticidal effective dose.

20. kill the method for coleopteran pest, and comprise described insect is contacted with the polypeptide as claimed in claim 12 of insecticidal effective dose, or to the polypeptide as claimed in claim 12 of described insect feeding insecticidal effective dose.

21. produce the method for the polypeptide with insecticidal activity, comprise the host cell that comprises DNA construct claimed in claim 3 is cultivated under the condition of nucleic acid molecule of expressing coding said polypeptide, described polypeptide is by the nucleotide sequence coded polypeptide of SEQ ID NO:1.

22. produce the method for plant, and described plant makes DNA construct stable integration to its genome, and described DNA construct comprises the nucleotide sequence that coding has the albumen of insecticidal activity, and wherein said nucleotide sequence is selected from:

A) nucleic acid molecule being formed by SEQ ID NO:1 nucleotide sequence; With

The nucleic acid molecule of the polypeptide that c) coding SEQ ID NO:2 aminoacid sequence forms;

Wherein said nucleotide sequence is operatively connected with the promotor that drives encoding sequence to express in vegetable cell.

23. methods as claimed in claim 22, wherein said plant is vegetable cell.

24. protective plants avoid the method for insect, comprise that the expression vector of the nucleotide sequence that at least one is comprised to coded insect-killing polypeptide is introduced described plant or its cell, and wherein said nucleotide sequence is selected from:

A) nucleic acid molecule being formed by SEQ ID NO:1 nucleotide sequence; With

The nucleic acid molecule of the polypeptide that c) coding SEQ ID NO:2 aminoacid sequence forms.

25. methods as claimed in claim 24, wherein said plant produces has the insecticidal peptide for the insecticidal activity of coleopteran pest.